Novel peptides and combination of peptides for use in immunotherapy against hepatocellular carcinoma (hcc) and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. In particular, the present invention relates to several novel peptide sequences and their variants derived from HLA class I and class II molecules of human tumor cells that can be used in vaccine compositions for eliciting anti-tumor immune responses or as targets for the development of pharmaceutically/immunologically active compounds and cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. 62/096,165, filed Dec. 23,2014, GB 1423016.3, filed Dec. 23, 2014 and GB 1501017.6, filed Jan. 21,2015, the content of which are incorporated herein by reference in theirentireties.

BACKGROUND

1. Field of the Invention

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules. In particular, the present invention relates toseveral novel peptide sequences and their variants derived from HLAclass I and class II molecules of human tumor cells that can be used invaccine compositions for eliciting anti-tumor immune responses or astargets for the development of pharmaceutically/immunologically activecompounds and cells.

2. Description of Related Art

Hepatocellular carcinoma (HCC) is one of the most common tumors in theworld and accounts for about 6% of all new cancer cases diagnosedworldwide. In 2012 about 782,000 new cases of HCC occurred in the world,making it the fifth most common cancer in men (554,000 cases) and theninth in women (228,000 cases) (http://globocan.iarc.fr). HCC is themost common primary liver malignancy accounting for over 80% of alladult primary liver cancers.

The distribution of HCC varies geographically, and rates of incidencedepend on gender. The age-standardized incidence rate (ASR) of HCC inmen is highest in Eastern Asia (31.9) and South-Eastern Asia (22.2),intermediate in Southern Europe (9.5) and Northern America (9.3) andlowest in Northern Europe (4.6) and South-Central Asia (3.7). Incidentrates of HCC in women are lower than male ASRs. The highest ASR in womenoccurs in Eastern Asia (10.2) and Western Africa (8.1), the lowest inNorthern Europe (1.9) and Micronesia (1.6).

The overall prognosis for patients with HCC is poor. The 5-year relativesurvival rate (5Y-RSR) from HCC is about 15%, depending on the stage atthe time of diagnosis. For localized HCC, where the cancer is stillconfined to the liver, the 5Y-RSR is about 28%. For regional and distantHCC, were the cancer has grown into nearby or distant organs, 5Y-RSRsare 7% and 2%, respectively.

The incidence of HCC is related to several risk factors, cirrhosis beingthe most important one. Cirrhosis often occurs alongside alcohol abuseor HBV or HCV infections, but can also be caused by metabolic diseaseslike type II diabetes. As a result, healthy liver tissue gets replacedby scar tissue, which increases the risk of cancer development.

Disease management depends on the tumor stage at the time of diagnosisand the overall condition of the liver. If possible, parts of the liver(partial hepatectomy) or the whole organ (liver resection) is removed bysurgery. Especially patients with small or completely resectable tumorsare qualified to receive a liver transplant.

If surgery is not a treatment option, different other therapies areavailable at hand. For tumor ablation, a probe is injected into theliver and the tumor is destroyed by radio or microwaves or cryotherapy.In embolization procedures, the blood supply of the tumor is blocked bymechanical or chemical means. High energy radio waves can be used todestroy the tumor in radiation therapy.

Chemotherapy against HCC includes combinations of doxorubicin,5-fluorouracil and cisplatin for systemic therapy and doxorubicin,floxuridine and mitomycin C for hepatic artery infusions. However, mostHCC show a high resistance to chemotherapeutics (Enguita-German andFortes, 2014).

Therapeutic options in advanced non-resectable HCC are limited toSorafenib, a multi-tyrosine kinase inhibitor (Chang et al., 2007;Wilhelm et al., 2004). Sorafenib is the only systemic drug confirmed toincrease survival by about 3 months and currently represents the onlyexperimental treatment option for such patients (Chapiro et al., 2014;Llovet et al., 2008).

Lately, a limited number of immunotherapy trials for HCC have beenconducted. Cytokines have been used to activate subsets of immune cellsand/or increase the tumor immunogenicity (Reinisch et al., 2002; Sangroet al., 2004). Other trials have focused on the infusion ofTumor-infiltrating lymphocytes or activated peripheral blood lymphocytes(Shi et al., 2004a; Takayama et al., 1991; Takayama et al., 2000).

So far, a small number of therapeutic vaccination trials have beenexecuted. Butterfield et al. conducted two trials using peptides derivedfrom alpha-fetoprotein (AFP) as a vaccine or DCs loaded with AFPpeptides ex vivo (Butterfield et al., 2003; Butterfield et al., 2006).In two different studies, autologous dendritic cells (DCs) were pulsedex vivo with autologous tumor lysate (Lee et al., 2005) or lysate of thehepatoblastoma cell line HepG2 (Palmer et al., 2009). So far,vaccination trials have only shown limited improvements in clinicaloutcomes.

SUMMARY

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 300 or a variant sequencethereof which is at least 80%, preferably at least 90%, homologous(preferably at least 80% or at least 90% identical) to SEQ ID NO: 1 toSEQ ID NO: 300, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 300 or a variant thereof, whichis at least 80%, preferably at least 88%, homologous (preferably atleast 80% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 300,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A through 1M show the over-presentation of various peptides innormal tissues (dark gray) and HCC (light gray). FIG. 1A: APOB, Peptide:ALVDTLKFV (A*02) (SEQ ID NO:7). FIG. 1B: ALDH1 L1, Peptide: KLQAGTVFV(A*02) (SEQ. ID NO:2). FIG. 1C: C8B, Peptide: AYLLQPSQF (A*24) (SEQ IDNO:200). FIG. 1D: FIG. 1D) RAD23B Peptide: KIDEKNFVV (SEQ ID NO:63).FIG. 1E: RAD23B Peptide: KIDEKNFVV (SEQ ID NO:63). FIG. 1F: RFNGPeptide: RLPPDTLLQQV (SEQ ID NO:92). FIG. 1G: RFNG Peptide: RLPPDTLLQQV(SEQ ID NO:92). FIG. 1H) FLVCR1 Peptide: SVWFGPKEV (SEQ ID NO:104). FIG.1I: FLVCR1 Peptide: SVWFGPKEV (SEQ ID NO:104). FIG. 1J: IKBKAP Peptide:LLFPHPVNQV (SEQ ID NO:156). FIG. 1K: IKBKAP Peptide: LLFPHPVNQV (SEQ IDNO: 156). FIG. 1L: NKD1 Peptide: FLDTPIAKV (SEQ ID NO:47). FIG. 1M: NKD1Peptide: FLDTPIAKV (SEQ ID NO:47).

FIGS. 2A through 2F show exemplary expression profiles (relativeexpression compared to normal kidney) of source genes of the presentinvention that are highly over-expressed or exclusively expressed in HCCin a panel of normal tissues (dark gray) and 12 HCC samples (gray). FIG.2A: APOB; FIG. 2B: AMACR; FIG. 2C: ALDH1L1; FIG. 2D: FGG; FIG. 2E: C8B;FIG. 2F: HSD17B6.

FIG. 3 shows exemplary flow cytometry results after peptide-specificmultimer staining.

FIG. 4 shows exemplary flow cytometry results after peptide-specificmultimer staining. CD8+ T cells were primed using artificial APCs coatedwith anti-CD28 mAb and HLAA*24 in complex with IMA-KLHL24-001 (Seq ID No190) peptide (4A, right panel) or IMAAPOB-006 (4B, right panel, Seq IDNo 218). Left panels (4A and 4B) show control staining.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 bind toHLA-A*02, peptides in Table 2 bind to HLA-A*24 alleles. The peptides inTable 3 have been disclosed before in large listings as results ofhigh-throughput screenings with high error rates or calculated usingalgorithms, but have not been associated with cancer at all before. Theybind to HLA-A*02. The peptides in Table 4 are additional peptides thatmay be useful in combination with the other peptides of the invention.Peptides bind A*02 or, where indicated, A*24. The peptides in Table 5are furthermore useful in the diagnosis and/or treatment of variousmalignancies that involve an over-expression or over-presentation of therespective underlying polypeptide.

TABLE 1 HLA-A*02 peptides according to the present  invention-S* =phosphoserine SEQ ID Official Gene No. Sequence GeneID(s) Symbol(s)   1VMAPFTMTI 338 APOB   2 KLQAGTVFV 10840 ALDH1L1   3 ILDDNMQKL 79611 ACSS3  4 KLQDFSDQL 338 APOB   5 ALVEQGFTV 338 APOB   6 KLSPTVVGL 8313 AXIN2  7 ALVDTLKFV 338 APOB   8 KLLEEATISV 54808 DYM   9 ALANQKLYSV 23195MDN1  10 SLLEEFDFHV 8615 USO1  11 SLSQELVGV 24149 ZNF318  12 FLAELAYDL2719 GPC3  13 GLIDTETAMKAV 3290 HSD11B1  14 ALADLTGTVV 23385 NCSTN  15LLYGHTVTV 347734 SLC35B2  16 SLLGGNIRL 2181 ACSL3  17 RVAS*PTSGV 8660IRS2  18 ALYGKTEVV 57513 CASKIN2  19 FLEETKATV 338 APOB  20 KLSNVLQQV338 APOB  21 QLIEVSSPITL 338 APOB  22 RIAGIRGIQGV 23167 EFR3A  23RLYDPASGTISL 23456 ABCB10  24 SLAEEKLQASV 2194 FASN  25 SLDGKAALTEL 338APOB  26 SLLHTIYEV 85407 NKD1  27 TLPDFRLPEI 338 APOB  28 TLQDHLNSL 338APOB  29 YIQDEINTI 338 APOB  30 YLGEGPRMV 5704 PSMC4  31 YQMDIQQEL 338APOB  32 ALNAVRLLV 9368 SLC9A3R1  33 LLHGHIVEL 57678 GPAM  34 SLAEGTATV540 ATP7B  35 SLQESILAQV 23644 EDC4  36 ILNVDGLIGV 47 ACLY  37LLLPLLPPLSP 347252 IGFBPL1  38 ALADVVHEA 26873 OPLAH  39 ALDPKANFST10188 TNK2  40 ALLAEGITVVV 54499 TMCO1  41 ALLELDEPLVL 2158 F9  42ALLGGNVRMML 2182 ACSL4  43 ALLGVVVTSV 444 ASPH  44 ALQDAIRQL 51268 PIPOX 45 ALQDQLVLV 183 AGT  46 AMAEMKVVL 11283, 4051, CYP4F8, 57834, 66002,CYP4F3,CYP4F11, 8529 CYP4F12,CYP4F2  47 FLDTPIAKV 85407 NKD1  48FLLEQPEIQV 5345 SERPINF2  49 FLYPEKDEPT 338 APOB  50 FTIPKLYQL 338 APOB 51 GLAEELVRA 5091 PC  52 GLFNAELLEA 3929 LBP  53 GLIHLEGDTV 81494 CFHR5 54 GLLDPNVKSIFV 79033 ERI3  55 GLYGRTIEL 55908 C19orf80  56 GVLPGLVGV162515 SLC16A11  57 HLTEAIQYV 6097 RORC  58 ILADLNLSV 55705 IPO9  59ILADTFIGV 222223 KIAA1324L  60 ILSPLSVAL 5345 SERPINF2  61 KIADFELPTI338 APOB  62 KIAGTNAEV 2752 GLUL  63 KIDEKNFVV 5887 RAD23B  64 KILEETLYV8443 GNPAT  65 KLFSGDELLEV 8777 MPDZ  66 KLHEEIDRV 1571 CYP2E1  67KLKETIQKL 338 APOB  68 KLLAATVLLL 336 APOA2  69 KLLDEVTYLEA 1573 CYP2J2 70 KLLDLETERILL 2803 GOLGA4  71 KLLDNWDSV 335 APOA1  72 KLSEAVTSV 55258THNSL2  73 KLTLVIISV 8647 ABCB11  74 KLYDLELIV 570 BAAT  75 KQMEPLHAV284111 SLC13A5  76 LLADIGGDPFAA 3268 AGFG2  77 LLHEENFSV 6942 TCF20  78LLIDDEYKV 23065 EMC1  79 LLLSTGYEA 23556 PIGN  80 LLYEGKLTL 440107PLEKHG7  81 NLASFIEQVAV 5092 PCBD1  82 NVFDGLVRV 338 APOB  83 QLHDFVMSL8647 ABCB11  84 QLTPVLVSV 1244 ABCC2  85 RILPKVLEV 10840 ALDH1L1  86RLAAFYSQV 91289 LMF2  87 RLFEENDVNL 5053 PAH  88 RLIDRIKTV 60560 NAA35 89 RLIEEIKNV 347051 SLC10A5  90 RLLDVLAPLV 80781 COL18A1  91 RLPDIPLRQV55656 INTS8  92 RLPPDTLLQQV 5986 RFNG  93 RLYTMDGITV 1571 CYP2E1  94RMSDVVKGV 113251 LARP4  95 SICNGVPMV 54575, 54576, UGT1A10,UGT1A8,  54577, 54578, UGT1A7,UGT1A6, 54579, 54600, UGT1A5,UGT1A9, 54657, 54658, UGT1A4,UGT1A1, 54659 UGT1A3  96 SLLEEPNVIRV 4703 NEB  97 SLLPQLIEV 338APOB  98 SLLSPEHLQYL 7512 XPNPEP2  99 SLSAFLPSL 54757 FAM20A 100SLVGDIGNVNM 1401 CRP 101 SLWEGGVRGV 411 ARSB 102 SLWSVARGV 57678 GPAM103 SMGDHLWVA 2752 GLUL 104 SVWFGPKEV 28982 FLVCR1 105 SVYDGKLLI 5445PON2 106 TLAAIIHGA 5243 ABCB1 107 TLGQFYQEV 3700, 375346 ITIH4,TMEM110108 TLLKKISEA 84675 TRIM55 109 TLYALSHAV 338 APOB 110 TVGGSEILFEV 1401CRP 111 TVMDIDTSGTFNV 26063, 4833 DECR2,NME4 112 VLGEVKVGV 122622 ADSSL1113 VLMDKLVEL 338 APOB 114 VLSQVYSKV 338 APOB 115 VVLDDKDYFL 100292290,HSPE1 3336 116 VVVIPAISAV 1528 CYB5A 117 YAFPKSITV 6566 SLC16A1 118YLDDEKNWGL 5005 ORM2 119 YLDKNLTVSV 100293534,  C4A,C4B 720,721 120YLGEEYVKA 7018 TF 121 YLITGNLEKL 1314 COPA 122 YLSQAADGAKVL 2584 GALK1123 YLWDLDHGFAGV 832 CAPZB 124 LLIDVVTYL 338 APOB 125 ALYGRLEVV 23294ANKS1A 126 TLLDSPIKV 338 APOB 127 VLIGSNHSL 9919 SEC16A 128 GLAFSLNGV81502 HM13 129 SQADVIPAV 55034 MOCOS 130 ALDAGAVYTL 10840 ALDH1L1 131ALDSGAFQSV 55907 CMAS 132 ALHEEVVGV 1593 CYP27A1 133 ALLEMDARL 54512EXOSC4 134 ALLETNPYLL 1209 CLPTM1 135 ALLGKIEKV 2590 GALNT2 136ALLNQHYQV 2058 EPRS 137 ALPTVLVGV 5351 PLOD1 138 ALSQVTLLL 392636 AGMO139 ALSSKPAEV 256987 SERINC5 140 ALTSISAGV 392636 AGMO 141 AMGEKSFSV57720 GPR107 142 AVIGGLIYV 366 AQP9 143 FILPDSLPLDTL 6632 SNRPD1 144FIQLITGV 477, 478 ATP1A2,ATP1A3 145 FLIAEYFEHV 23743, 635 BHMT2,BHMT 146FLVVTEQAHTV 3953 LEPR 147 GLAPGGLAVV 58525 WIZ 148 GLFAPLVFL 6566SLC16A1 149 GLLSGLDIMEV 383 ARG1 150 GLSNLGIKSI 122553 TRAPPC6B 151HLAKVTAEV 6184 RPN1 152 KLDNNLDSV 80232 WDR26 153 KLIEVNEEL 100507203SMLR1 154 KLTDHLKYV 3250 HPR 155 LLEPYKPPSAQ 439 ASNA1 156 LLFPHPVNQV8518 IKBKAP 157 QLLPNLRAV 5092 PCBD1 158 RIISGLVKV 101060372, FMO5 2330159 RLFPDGIVTV 152831 KLB 160 RLLAKIICL 3075 CFH 161 RLLDEQFAV 9026HIP1R 162 RLMSALTQV 9462 RASAL2 163 RLTESVLYL 368 ABCC6 164 RMLIKLLEV6710, 6711 SPTB,SPTBN1 165 RVIEHVEQV 3034 HAL 166 SILDIVTKV 130132 RFTN2167 SLAESSFDV 54658 UGT1A1 168 SLAVLVPIV 1361 CPB2 169 SLFEWFHPL 2519FUCA2 170 SLHNGVIQL 1314 COPA 171 SLIPAVLTV 57462 KIAA1161 172 SLLNFLQHL2968 GTF2H4 173 SLTSEIHFL 55755 CDK5RAP2 174 TLAELGAVQV 2875 GPT 175TLFEHLPHI 2888 GRB14 176 TLGQIWDV 1778 DYNC1H1 177 VLDEPYEKV 100034743,PDZK1P2,PDZK1, 5174,   PDZK1P1 728939 178 YIFTTPKSV 22862 FNDC3A 179YIHNILYEV 160518 DENND5B 180 YLGPHIASVTL 81671 VMP1 181 YLLEKFVAV1663, 440081, DDX11,DDX12P 642846 182 YLLHFPMAL 1109 AKR1C4 183YLYNNEEQVGL 1109 AKR1C4 184 VVLDGGQIVTV 6506 SLC1A2 185 ALFPALRPGGFQA8878 SQSTM1 186 VLLAQIIQV 89797 NAV2

TABLE 2 HLA-A*24 peptides according to the present invention with SEQID numbers-S* = phosphoserine SEQ ID No. Sequence GeneID(s)Official Gene Symbol(s) 187 SYPTFFPRF 6596 HLTF 188 RYSAGWDAKF 8630HSD17B6 189 AFSPDSHYLLF 3679 ITGA7 190 RYNEKCFKL 54800 KLHL24 191KYPDIISRI 3978 LIG1 192 SYITKPEKW 79694 MANEA 193 IYPGAFVDL 51360 MBTPS2194 QYASRFVQL 10733 PLK4 195 RYAPPPSFSEF 29066 ZC3H7A 196 AYLKWISQI60561 RINT1 197 RWPKKSAEF 100132742, 100526842, RPL17P7, RPL17-6139, 645296, 645441 C18orf32, RPL17, RPL17P39, RPL17P6 198 LYWSHPRKF6235, 648343 RPS29, RPS29P9 199 KFVTVQATF 718 C3 200 AYLLQPSQF 732 C8B201 AYVNTFHNI 1201 CLN3 202 AYGTYRSNF 9919 SEC16A 203 YYGILQEKI 10237SLC35B1 204 KYRLTYAYF 2266 FGG 205 VYGLQRNLL 57159, 84675, 84676TRIM54, TRIM55, TRIM63 206 KWPETPLLL 55757 UGGT2 207 IYLERFPIF 51096UTP18 208 SYNPAENAVLL 1314 COPA 209 VFHPRQELI 1314 COPA 210 AYPAIRYLL7818 DAP3 211 IYIPSYFDF 27042 DIEXF 212 VYGDVISNI 8893 EIF2B5 213YYNKVSTVF 8661 EIF3A 214 IYVTSIEQI 55879 GABRQ 215 IYTGNISSF 8836 GGH216 IYADVGEEF 100302182, 11052 MIR1279, CPSF6 217 DYIPYVFKL 338 APOB 218VYQGAIRQI 338 APOB

TABLE 3 Additional peptides according to the present invention with no prior known cancer association-S* = phosphoserine SEQ ID  OfficialNo. Sequence GeneID(s) Gene Symbol(s) 219 GVMAGDIYSV 123 PLIN2 220SLLEKELESV 1819 DRG2 221 ALCEENMRGV 1938 EEF2 222 LTDITKGV 1938 EEF2 223FLFNTENKLLL 3422 ID11 224 ALASVIKEL 28981 IFT81 225 KMDPVAYRV 5859 QARS226 AVLGPLGLQEV 79178 THTPA 227 ALLKVNQEL 25813 SAMM50 228 YLITSVELL2182 ACSL4 229 KMFESFIESV 5576 PRKAR2A 230 VLTEFTREV 55705 IPO9 231RLFNDPVAMV 10195 ALG3 232 KLAEIVKQV 8550 MAPKAPK5 233 ALLGKLDAI 5876RABGGTB 234 YLEPYLKEV 727947,7381 UQCRB 235 KLFEEIREI 255394 TCP11L2 236ALADKELLPSV 84883 AIFM2 237 ALRGEIETV 10128 LRPPRC 238 AMPPPPPQGV 5885RAD21 239 FLLGFIPAKA 5976 UPF1 240 FLWERPTLLV 79922 MRM1 241 FVLPLLGLHEA55161 TMEM33 242 GLFAPVHKV 6249 CLIP1 243 GLLDNPELRV 26263 FBXO22 244KIAELLENV 9100 USP10 245 KLGAVFNQV 23450 SF3B3 246 KLISSYYNV 84928TMEM209 247 KLLDTMVDTFL 100527963,11243 PMF1-BGLAP,PMF1 248 KLNDLIQRL1314 COPA 249 LLLGERVAL 23475 QPRT 250 NLAEVVERV 26263 FBXO22 251RLFADILNDV 64755 C16orf58 252 RTIEYLEEV 3030 HADHA 253 RVPPPPQSV 6464SHC1 254 RVQEAIAEV 57678 GPAM 255 SLFGQDVKAV 26036 ZNF451 256SLFQGVEFHYV 3930 LBR 257 SLLEKAGPEL 54625 PARP14 258 SLMGPVVHEV 5116PCNT 259 TLITDGMRSV 29894 CPSF1 260 TLMDMRLSQV 24148 PRPF6 261VLFQEALWHV 2194 FASN 262 VLPNFLPYNV 10299 MARCH6 263 VLYPSLKEI50717,5824 DCAF8,PEX19 264 VMQDPEFLQSV 266971,5710 PIPSL, PSMD4 265WLIEDGKVVTV 10726 NUDC 266 SLLESNKDLLL 6520 SLC3A2 267 ALNENINQV 80025PANK2 268 KLYQEVEIASV 5976 UPF1 269 YLMEGSYNKV 5714 PSMD8 270 SVLDQKILL9875 URB1 271 LLLDKLILL 85440 DOCK7 272 QQLDSKFLEQV 6772 STAT1 273AILETAPKEV 6238 RRBP1 274 ALAEALKEV 55164 SHQ1 275 ALIEGAGILL 10440TIMM17A 276 ALLEADVNIKL 6729 SRP54 277 ALLEENSTPQL 83933 HDAC10 278ALTSVVVTL 1021 CDK6 279 ALVVTGMHTI 51479 ANKFY1 280 ATLNIIHSV 51542VPS54 281 GLLAGDRLVEV 9368 SLC9A3R1 282 GQFPSYLETV 54919 HEATR2 283ILSGIGVSQV 3703 STT3A 284 KLDAFVEGV 528 ATP6V1C1 285 KLLDLSDSTSV 6093ROCK1 286 KVLDKVFRA 375056 MIA3 287 LIGEFLEKV 8731 RNMT 288 LLDDSLVSI25824 PRDX5 289 LLLEEGGLVQV 7353 UFD1L 290 NLIDLDDLYV 57187 THOC2 291QLIDYERQL 11072 DUSP14 292 RIPAYFVTV 7407 VARS 293 FLASESLIKQI 4736RPL10A 294 RLIDLHTNV 23256 SCFD1 295 SLFSSPPEI 252983 STXBP4 296SLLSGRISTL 51133,92799 KCTD3,SHKBP1 297 TLFYSLREV 80233 C17orf70 298TMAKESSIIGV 1429 CRYZ 299 ALLRVTPFI 401505 TOMM5 300 TLAQQPTAV 4802 NFYC

TABLE 4 Peptides useful for e.g. personalized cancer therapies-S* = 32phosphoserine SEQ ID No. Sequence GeneID(s) Official Gene Symbol(s) 301VLADFGARV 114899,23600 C1QTNF3,AMACR 302 KIQEILTQV 10643 IGF2BP3 303GVYDGEEHSV 4113 MAGEB2 304 SLIDQFFGV 9097 USP14 305 GVLENIFGV 399909PCNXL3 306 KLVEFDFLGA 10460 TACC3 307 AVVEFLTSV 29102 DROSHA 308ALLRTVVSV 2590 GALNT2 309 GLIEIISNA 23020 SNRNP200 310 SLWGGDVVL 157680VPS13B 311 FLIPIYHQV 31 ACACA 312 RLGIKPESV 1466 CSRP2 313 LTAPPEALLMV79050 NOC4L 314 YLAPFLRNV 23019 CNOT1 315 KVLDGSPIEV 29974 A1CF 316LLREKVEFL 4779 NFE2L1 317 KLPEKWESV 26156 RSL1D1 318 KLNEINEKI 1373 CPS1319 KLFNEFIQL 10885 WDR3 320 GLADNTVIAKV 6897 TARS 321 GVIAEILRGV 10528NOP56 322 ILYDIPDIRL 10667 FARS2 323 KIIDEDGLLNL 5981 RFC1 324RLFETKITQV 100293534,720,721 C4A,C4B 325 RLSEAIVTV 51249 TMEM69 326ALSDGVHKI 55179 FAIM 327 GLNEEIARV 10403 NDC80 328 RLEEDDGDVAM 10482NXF1 329 SLIEDLILL 64754 SMYD3 330 SMSADVPLV 5111 PCNA 331 SLLAQNTSWLL7070 THY1 332 AMLAVLHTV 60673 C12orf44 333 GLAEDIDKGEV 1938 EEF2 334SILTIEDGIFEV 100287551,3306,3312 HSPA8P8,HSPA2,HSPA8 335 SLLPVDIRQYL6773 STAT2 336 YLPTFFLTV 54898 ELOVL2 337 TLLAAEFLKQV 100288772,10574CCT7P2,CCT7 338 KLFDSDPITVTV 1191 CLU 339 RLISKFDTV 1977 EIF4E 340KVFDEVIEV 8908 GYG2 341 YLAIGIHEL 3034 HAL 342 AMSSKFFLV 7474 WNT5A 343LLLPDYYLV 27044 SND1 344 VYISSLALL  10213 PSMD14 (A*24) 345 SYNPLWLRI 259266 ASPM (A*24) 346 LYQILQGIVF  983 CDK1 (A*24) 347 ALNPADITV 51497TH1L 348 AYKPGALTF 84883 AIFM2

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, pancreatic cancer, colonor rectal cancer, kidney cancer, brain cancer, and/or leukemias.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 300. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 124 (see Table 1), preferably for A*02binding, and from the group consisting of SEQ ID NO: 187 to SEQ ID NO:218 (see Table 2) preferably for A*24 binding, and their uses in theimmunotherapy of HCC, brain cancer, kidney cancer, pancreatic cancer,colon or rectal cancer or leukemia, and preferably HCC.

As shown in the following tables 5A and B, many of the peptidesaccording to the present invention can also be used in the immunotherapyof other indications. The tables show, for selected peptides on whichadditional tumor types they were found showing over-presentation(including specific presentation) on more than 5% of the measured tumorsamples, or presentation on more than 5% of the measured tumor sampleswith a ratio of geometric means tumor vs normal tissues being largerthan 3. Over-presentation is defined as higher presentation on the tumorsample as compared to the normal sample with highest presentation.Normal tissues against which over-presentation was tested were: adiposetissue, adrenal gland, blood cells, blood vessel, bone marrow, brain,cartilage, esophagus, eye, gallbladder, heart, kidney, large intestine,liver, lung, lymph node, nerve, pancreas, parathyroid gland, peritoneum,pituitary, pleura, salivary gland, skeletal muscle, skin, smallintestine, spleen, stomach, thyroid gland, trachea, ureter, urinarybladder.

TABLE 5A Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases-S* = phosphoserine SEQ ID Other relevant No.Sequence organs/diseases   1 VMAPFTMTI Pancreas   6 KLSPTVVGLColon, Rectum  10 SLLEEFDFHV Kidney  14 ALADLTGTVVKidney, Brain, Pancreas  15 LLYGHTVTVKidney, Brain, Colon, Rectum, Pancreas  16 SLLGGNIRLBrain, Colon, Rectum  17 RVAS*PTSGV Brain  22 RIAGIRGIQGVKidney, Colon, Rectum  26 SLLHTIYEV Colon, Rectum  30 YLGEGPRMVColon, Rectum, CLL  34 SLAEGTATV Colon, Rectum  36 ILNVDGLIGVKidney, Brain, Colon, Rectum  39 ALDPKANFST Kidney, Brain  41ALLELDEPLVL Pancreas  43 ALLGVWTSV Pancreas  47 FLDTPIAKVBrain, Colon, Rectum  51 GLAEELVRA Brain  54 GLLDPNVKSIFV Kidney, Brain 55 GLYGRTIEL Kidney  58 ILADLNLSV Pancreas  59 ILADTFIGVColon, Rectum, Pancreas  60 ILSPLSVAL Kidney, Pancreas  65 KLFSGDELLEVBrain, Colon, Rectum  69 KLLDEVTYLEA Colon, Rectum  70 KLLDLETERILLColon, Rectum  72 KLSEAVTSV Kidney  77 LLHEENFSV Kidney, Colon, Rectum 80 LLYEGKLTL Colon, Rectum  81 NLASFIEQVAVKidney, Colon, Rectum, Pancreas  88 RLIDRIKTV Brain, Colon, Rectum  90RLLDVLAPLV Kidney  96 SLLEEPNVIRV Kidney 101 SLWEGGVRGV Brain 112VLGEVKVGV Kidney 116 WVIPAISAV Kidney 119 YLDKNLTVSV Kidney 121YLITGNLEKL Kidney, Colon, Rectum, Pancreas 123 YLWDLDHGFAGVBrain, Colon, Rectum 125 ALYGRLEVV Brain, Colon, Rectum 127 VLIGSNHSLColon, Rectum 133 ALLEMDARL Kidney, Brain, Colon, Rectum 134 ALLETNPYLLBrain 135 ALLGKIEKV Brain, Pancreas 137 ALPTVLVGVKidney, Brain, Colon, Rectum 138 ALSQVTLLL Kidney 139 ALSSKPAEVColon, Rectum, Pancreas 141 AMGEKSFSV Brain 144 FIQLITGV Pancreas 147GLAPGGLAVV Brain 148 GLFAPLVFL Kidney 161 RLLDEQFAV Brain 166 SILDIVTKVBrain 169 SLFEWFHPL Kidney, Brain, Colon, Rectum 170 SLHNGVIQL Kidney172 SLLNFLQHL Kidney, Colon, Rectum, CLL 173 SLTSEIHFL CLL 176 TLGQIWDVBrain, Colon, Rectum, Pancreas 177 VLDEPYEKV Kidney 179 YIHNILYEV Brain181 YLLEKFVAV Colon, Rectum 184 WLDGGQIVTV Brain 186 VLLAQIIQVKidney, Brain, Colon, Rectum 187 SYPTFFPRF Kidney, Brain 189 AFSPDSHYLLFKidney, Brain 191 KYPDIISRI Brain 192 SYITKPEKW Kidney, Brain 193IYPGAFVDL Brain 194 QYASRFVQL Brain 195 RYAPPPSFSEF Brain 196 AYLKWISQIBrain 197 RWPKKSAEF Kidney, Brain 198 LYWSHPRKF Kidney 199 KFVTVQATFBrain 203 YYGILQEKI Kidney, Brain 206 KWPETPLLL Kidney, Brain 208SYNPAENAVLL Brain 214 IYVTSIEQI Brain 219 GVMAGDIYSV Kidney 220SLLEKELESV Brain 221 ALCEENMRGV Kidney, Brain, Colon, Rectum 223FLFNTENKLLL Colon, Rectum 224 ALASVIKEL Brain 229 KMFESFIESVKidney, Brain, Colon, Rectum 230 VLTEFTREV Kidney, Brain, Colon, Rectum231 RLFNDPVAMV Brain, Colon, Rectum 232 KLAEIVKQV Colon, Rectum 233ALLGKLDAI Kidney, Colon, Rectum 234 YLEPYLKEVKidney, Brain, Colon, Rectum 236 ALADKELLPSVKidney, Colon, Rectum, Pancreas 237 ALRGEIETV Colon, Rectum 238AMPPPPPQGV Brain, Colon, Rectum 239 FLLGFIPAKA Brain 240 FLWERPTLLV CLL244 KIAELLENV Brain, Colon, Rectum 245 KLGAVFNQV Brain 247 KLLDTMVDTFLColon, Rectum 248 KLNDLIQRL Pancreas 249 LLLGERVAL Colon, Rectum 250NLAEVVERV Brain, Colon, Rectum, CLL 251 RLFADILNDV Brain, Colon, Rectum255 SLFGQDVKAV Kidney, Brain, Colon, Rectum 258 SLMGPVVHEV Brain 259TLITDGMRSV Brain 260 TLMDMRLSQV Kidney, Brain, Colon, Rectum 261VLFQEALWHV Colon, Rectum 266 SLLESNKDLLL Colon, Rectum 268 KLYQEVEIASVBrain 269 YLMEGSYNKV Brain, Colon, Rectum 270 SVLDQKILL Kidney, Brain271 LLLDKLILL Brain, Colon, Rectum 272 QQLDSKFLEQV Kidney, Brain 274ALAEALKEV Colon, Rectum 275 ALIEGAGILL Kidney, Colon, Rectum, Pancreas276 ALLEADVNIKL Pancreas 277 ALLEENSTPQL Kidney 278 ALTSVVVTLKidney, Brain 279 ALWTGMHTI Kidney, Brain 281 GLLAGDRLVEV Kidney 282GQFPSYLETV Kidney, Brain, Colon, Rectum 283 ILSGIGVSQV Pancreas 285KLLDLSDSTSV Kidney, Colon, Rectum 286 KVLDKVFRA Pancreas 287 LIGEFLEKVCLL 288 LLDDSLVSI Pancreas 289 LLLEEGGLVQVKidney, Colon, Rectum, Pancreas 290 NLIDLDDLYVBrain, Colon, Rectum, Pancreas 291 QLIDYERQLKidney, Colon, Rectum, Pancreas 292 RIPAYFVTV Kidney 293 FLASESLIKQIBrain, Colon, Rectum 295 SLFSSPPEI Kidney, Brain 296 SLLSGRISTL Kidney297 TLFYSLREV Kidney, Brain, Colon, Rectum 299 ALLRVTPFI CLL 300TLAQQPTAV Pancreas 301 VLADFGARV Kidney, Colon, Rectum 302 KIQEILTQVKidney, Brain, Colon, Rectum,  Pancreas, CLL 304 SLIDQFFGVBrain, Colon, Rectum, Pancreas 305 GVLENIFGV Kidney, Brain 306KLVEFDFLGA Brain, Colon, Rectum 308 ALLRTVVSV Kidney, Pancreas 309GLIEIISNA Brain 310 SLWGGDVVL Brain, Colon, Rectum 311 FLIPIYHQVKidney, Brain 312 RLGIKPESV Brain 313 LTAPPEALLMVKidney, Brain, Colon, Rectum, Pancreas 315 KVLDGSPIEV Kidney 316LLREKVEFL Kidney, Brain, Colon, Rectum, Pancreas 317 KLPEKWESVBrain, Colon, Rectum, Pancreas 319 KLFNEFIQLKidney, Brain, Colon, Rectum 321 GVIAEILRGV Kidney, Brain 324 RLFETKITQVKidney 325 RLSEAIVTV Brain, Pancreas 326 ALSDGVHKI Pancreas 327GLNEEIARV Brain, Colon, Rectum 328 RLEEDDGDVAMKidney, Brain, Colon, Rectum 329 SLIEDLILLKidney, Brain, Colon, Rectum, Pancreas 330 SMSADVPLVBrain, Colon, Rectum 331 SLLAQNTSWLL Brain, Colon, Rectum, Pancreas 332AMLAVLHTV Brain, Colon, Rectum 333 GLAEDIDKGEV Kidney, Brain 334SILTIEDGIFEV Kidney, Brain, Colon, Rectum,  Pancreas, CLL 335SLLPVDIRQYL Kidney, CLL 336 YLPTFFLTV Kidney, Brain 337 TLLAAEFLKQVBrain 338 KLFDSDPITVTV Brain 339 RLISKFDTV Brain 340 KVFDEVIEV Brain 342AMSSKFFLV Brain, Colon, Rectum, Pancreas 343 LLLPDYYLV Brain, Pancreas344 VYISSLALL  Brain (A*24) 345 SYNPLWLRI Brain (A*24) 346 LYQILQGIVFKidney (A*24) 347 ALNPADITV Brain

TABLE 5BPeptides according to the present invention and their specific uses inother proliferative diseases, especially in other cancerous diseases- S* = phosphoserine SEQ ID NO. Sequence Additional Entities 189AFSPDSHYLLF NSCLC, PrC 273 AILETAPKEV Esophageal Cancer 236 ALADKELLPSVNSCLC, SCLC, GC, Melanoma, OC, EsophagealCancer, Urinary bladder cancer, Gallbladder Cancer,Bile Duct Cancer, NHL  14 ALADLTGTWNSCLC, SCLC, BRCA, OC, Esophageal Cancer,Urinary bladder cancer, Uterine Cancer, PC  38 ALADVVHEA BRCA, OC 274ALAEALKEV BRCA, MCC, Melanoma, OC, Uterine Cancer, AML   9 ALANQKLYSVNSCLC, CRC, MCC, OC, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer, PC 224 ALASVIKELSCLC, PC, Melanoma 221 ALCEENMRGV NSCLC, SCLC, MCC, Melanoma 131ALDSGAFQSV CRC, Melanoma, Gallbladder Cancer, Bile Duct Cancer 185ALFPALRPGGF Gallbladder Cancer, Bile Duct Cancer QA 275 ALIEGAGILLSCLC, MCC, Melanoma, OC, Esophageal Cancer,Urinary bladder cancer, Uterine Cancer, NHL 276 ALLEADVNIKLGallbladder Cancer, Bile Duct Cancer, OC 277 ALLEENSTPQLSCLC, CLL, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, NHL133 ALLEMDARL NSCLC, SCLC, BRCA, MCC, Melanoma, OC,Esophageal Cancer, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL 134 ALLETNPYLLUrinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer 135ALLGKIEKV NSCLC, SCLC, BRCA, OC, Gallbladder Cancer, Bile Duct Cancer233 ALLGKLDAI CLL, OC, Urinary bladder cancer, GallbladderCancer, Bile Duct Cancer, AML, NHL  43 ALLGVWTSVSCLC, Brain Cancer, CLL, BRCA, PC 227 ALLKVNQEL Melanoma, Uterine Cancer136 ALLNQHYQV BRCA, OC 299 ALLRVTPFI NHL, OC  32 ALNAVRLLV SCLC 267ALNENINQV SCLC, Brain Cancer, MCC, Melanoma, EsophagealCancer, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer 137ALPTVLVGV NSCLC, GC, BRCA, Melanoma, Esophageal Cancer,Urinary bladder cancer, Uterine Cancer, GallbladderCancer, Bile Duct Cancer, PC  45 ALQDQLVLV SCLC, Brain Cancer 237ALRGEIETV SCLC, BRCA, OC, Esophageal Cancer, Urinary bladder cancer, NHL139 ALSSKPAEV PrC, OC, Uterine Cancer 278 ALTSVVVTLNSCLC, SCLC, GC, Esophageal Cancer, Urinarybladder cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL 279ALWTGMHTI Esophageal Cancer, Urinary bladder cancer, PC 125 ALYGRLEVVBRCA, MCC, Melanoma, OC, Urinary bladder cancer, Uterine Cancer 141AMGEKSFSV Melanoma 238 AMPPPPPQGV NSCLC, SCLC, BRCA, MCC, Melanoma, OC,Esophageal Cancer, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer, NHL 342 AMSSKFFLVNSCLC, GC, PrC, BRCA, Melanoma, OC,Esophageal Cancer, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer, PC 280 ATLNIIHSVUrinary bladder cancer 226 AVLGPLGLQEV PrC, Melanoma, OC 202 AYGTYRSNFNSCLC 196 AYLKWISQI NSCLC 210 AYPAIRYLL NSCLC, GC 144 FIQLITGVNSCLC, Brain Cancer, Urinary bladder cancer 293 FLASESLIKQINSCLC, PrC, MCC, Melanoma, OC, EsophagealCancer, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer, NHL, PC  47 FLDTPIAKVNSCLC, GC, Esophageal Cancer 223 FLFNTENKLLLMelanoma, Urinary bladder cancer, NHL 145 FLIAEYFEHV SCLC 239 FLLGFIPAKAUrinary bladder cancer, AML, NHL 128 GLAFSLNGVMelanoma, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 147GLAPGGLAVV NSCLC, SCLC, PrC, BRCA, Melanoma, OC,Esophageal Cancer, Urinary bladder cancer, Uterine Cancer 148 GLFAPLVFLEsophageal Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 242GLFAPVHKV Urinary bladder cancer  52 GLFNAELLEA SCLC 281 GLLAGDRLVEVNSCLC, SCLC, OC, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer, NHL 243 GLLDNPELRVUrinary bladder cancer  54 GLLDPNVKSIFVNSCLC, SCLC, OC, Urinary bladder cancer 149 GLLSGLDIMEV SCLC 150GLSNLGIKSI Urinary bladder cancer, NHL  55 GLYGRTIEL SCLC 282 GQFPSYLETVNSCLC, CLL, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, NHL219 GVMAGDIYSV SCLC, Gallbladder Cancer, Bile Duct Cancer, PC 151HLAKVTAEV SCLC, OC, Urinary bladder cancer  57 HLTEAIQYV NHL  58ILADLNLSV BRCA  59 ILADTFIGVNSCLC, SCLC, GC, OC, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer, NHL  36 ILNVDGLIGVNSCLC, SCLC, PrC, Melanoma, OC, Urinary bladdercancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL, PC283 ILSGIGVSQV NSCLC, PrC, BRCA, Melanoma, OC, EsophagealCancer, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer, NHL, PC 216 IYADVGEEFNSCLC, GC, PrC 211 IYIPSYFDF NSCLC, Brain Cancer, GC 207 IYLERFPIFNSCLC, GC 193 IYPGAFVDL NSCLC 214 IYVTSIEQI NSCLC, RCC 244 KIAELLENVNSCLC, SCLC, PrC, CLL, BRCA, Melanoma, OC, Uterine Cancer, AML, NHL  62KIAGTNAEV BRCA  63 KIDEKNFVVSCLC, Brain Cancer, Urinary bladder cancer, Uterine Cancer  64 KILEETLYVEsophageal Cancer, Urinary bladder cancer, Uterine Cancer 302 KIQEILTQVNSCLC, SCLC, GC, Melanoma, OC, EsophagealCancer, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer, AML, NHL, PC 232 KLAEIVKQVNSCLC, SCLC, BRCA, Melanoma, OC, Urinary bladder cancer 284 KLDAFVEGVBRCA, OC 152 KLDNNLDSV BRCA, Melanoma 235 KLFEEIREINSCLC, CRC, Melanoma, Urinary bladder cancer 245 KLGAVFNQVNSCLC, SCLC, RCC, PrC, BRCA, Melanoma,Esophageal Cancer, Urinary bladder cancer, Uterine Cancer 246 KLISSYYNVOC  69 KLLDEVTYLEA Urinary bladder cancer 70 KLLDLETERILLOC, Uterine Cancer 285 KLLDLSDSTSV SCLC, Uterine Cancer 247 KLLDTMVDTFLNSCLC, SCLC, RCC, Brain Cancer, CLL, BRCA, OC,Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, AML, NHL   8KLLEEATISV SCLC, MCC, Melanoma, Urinary bladder cancer, Uterine Cancer248 KLNDLIQRL GC, Uterine Cancer  20 KLSNVLQQV SCLC   6 KLSPTWGL CLL, OC154 KLTDHLKYV SCLC 268 KLYQEVEIASVCRC, MCC, Melanoma, Urinary bladder cancer 225 KMDPVAYRVCRC, PrC, BRCA, Urinary bladder cancer, Uterine Cancer 229 KMFESFIESVNSCLC, SCLC, PrC, OC, Urinary bladder cancer 286 KVLDKVFRACRC, Gallbladder Cancer, Bile Duct Cancer 206 KWPETPLLL GC 191 KYPDIISRINSCLC, GC 287 LIGEFLEKV SCLC, RCC, Melanoma, OC, Esophageal Cancer,Urinary bladder cancer, NHL 288 LLDDSLVSIMelanoma, Urinary bladder cancer, AML 156 LLFPHPVNQVNSCLC, SCLC, OC, Esophageal Cancer, Urinary bladder cancer  77 LLHEENFSVNSCLC, SCLC, OC, Esophageal Cancer, Urinarybladder cancer, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer, NHL  78 LLIDDEYKVEsophageal Cancer, Urinary bladder cancer, PC 271 LLLDKLILLCLL, Melanoma, OC, Urinary bladder cancer, Uterine Cancer 289LLLEEGGLVQV NSCLC, SCLC, PrC, Melanoma, OC, Urinary bladder cancer, NHL249 LLLGERVAL OC  37 LLLPLLPPLSPSCLC, PC, MCC, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer  80LLYEGKLTL OC  15 LLYGHTVTV NSCLC, SCLC, BRCA, Melanoma, OC, EsophagealCancer, Urinary bladder cancer, NHL, PC 222 LTDITKGV BRCA, NHL 346LYQILQGIVF NSCLC, Brain Cancer, GC 198 LYWSHPRKF NSCLC 250 NLAEVVERVNSCLC, SCLC, PrC, BRCA, MCC, OC, Gallbladder Cancer, Bile Duct Cancer 81 NLASFIEQVAV SCLC, PrC, OC, Uterine Cancer, NHL 290 NLIDLDDLYVSCLC, PrC, MCC, OC, Urinary bladder cancer, Uterine Cancer 291 QLIDYERQLNSCLC, SCLC, Brain Cancer, BRCA, Melanoma,Esophageal Cancer, Urinary bladder cancer, NHL, PC 157 QLLPNLRAV RCC 272QQLDSKFLEQV MCC, OC, NHL 194 QYASRFVQL NSCLC, GC  22 RIAGIRGIQGVNSCLC, PrC, BRCA, OC, NHL 292 RIPAYFVTV GC, BRCA, Melanoma, NHL  86RLAAFYSQV AML 251 RLFADILNDV NSCLC, SCLC, PrC, Melanoma, Urinary bladdercancer, Uterine Cancer 231 RLFNDPVAMVNSCLC, SCLC, MCC, Melanoma, OC, Urinarybladder cancer, Gallbladder Cancer, Bile Duct Cancer 294 RLIDLHTNVEsophageal Cancer  88 RLIDRIKTV NSCLC, SCLC, OC, AML, NHL  89 RLIEEIKNVSCLC 161 RLLDEQFAV SCLC, BRCA  90 RLLDVLAPLV BRCA  91 RLPDIPLRQVNSCLC, CLL, Urinary bladder cancer, NHL  92 RLPPDTLLQQVUrinary bladder cancer  23 RLYDPASGTISL CLL, Melanoma, NHL 164 RMLIKLLEVSCLC, CRC  94 RMSDWKGV BRCA, OC 252 RTIEYLEEV Melanoma  17 RVAJPTSGV AML197 RWPKKSAEF NSCLC 195 RYAPPPSFSEF NSCLC 190 RYNEKCFKL NSCLC  95SICNGVPMV Urinary bladder cancer 166 SILDIVTKVSCLC, CLL, MCC, Melanoma, Urinary bladder cancer, AML  24 SLAEEKLQASVPrC, BRCA, Urinary bladder cancer 169 SLFEWFHPLNSCLC, Gallbladder Cancer, Bile Duct Cancer 255 SLFGQDVKAVNSCLC, SCLC, CLL, BRCA, MCC, Melanoma, OC,Esophageal Cancer, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer, NHL, PC 256 SLFQGVEFHYVCRC, CLL, MCC, NHL 295 SLFSSPPEINSCLC, SCLC, CRC, PrC, BRCA, Melanoma, OC,Urinary bladder cancer, Uterine Cancer 170 SLHNGVIQLNSCLC, Urinary bladder cancer, Uterine Cancer, NHL 331 SLLAQNTSWLLNSCLC, RCC, GC, BRCA, Melanoma, EsophagealCancer, Urinary bladder cancer  10 SLLEEFDFHVNSCLC, BRCA, OC, Esophageal Cancer, Uterine Cancer  96 SLLEEPNVIRVMelanoma, Gallbladder Cancer, Bile Duct Cancer 257 SLLEKAGPELCLL, Melanoma, OC 220 SLLEKELESVNSCLC, SCLC, PrC, CLL, BRCA, OC, EsophagealCancer, Urinary bladder cancer, NHL 266 SLLESNKDLLLSCLC, MCC, Uterine Cancer  16 SLLGGNIRLGC, PrC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, PC 26 SLLHTIYEV PrC, BRCA, Esophageal Cancer, NHL 172 SLLNFLQHL AML  97SLLPQLIEV SCLC 296 SLLSGRISTL BRCA, Urinary bladder cancer 258SLMGPVVHEV SCLC, MCC, Melanoma, OC, Urinary bladder cancer, NHL  35SLQESILAQV NSCLC, SCLC, PrC, MCC, Melanoma, Urinary bladder cancer, AML 99 SLSAFLPSL OC, Esophageal Cancer, Gallbladder Cancer, BileDuct Cancer, NHL  11 SLSQELVGVBrain Cancer, Melanoma, Uterine Cancer, NHL 173 SLTSEIHFLUterine Cancer, NHL 101 SLWEGGVRGV Melanoma 103 SMGDHLWVABRCA, Urinary bladder cancer, NHL 330 SMSADVPLVNSCLC, SCLC, BRCA, MCC, Melanoma, EsophagealCancer, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer, AML, NHL 129 SQADVIPAVUrinary bladder cancer 270 SVLDQKILLMelanoma, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer, NHL 104 SVWFGPKEVSCLC, Melanoma, Urinary bladder cancer, PC 192 SYITKPEKW NSCLC 208SYNPAENAVLL NSCLC 345 SYNPLWLRI NSCLC, RCC, GC 187 SYPTFFPRF NSCLC, PrC300 TLAQQPTAV Urinary bladder cancer, Uterine Cancer, GallbladderCancer, Bile Duct Cancer, NHL 297 TLFYSLREV BRCA, Uterine Cancer, AML176 TLGQIWDV NSCLC, GC, Melanoma, Urinary bladder cancer, PC 259TLITDGMRSV RCC, CRC, OC, Esophageal Cancer, Urinary bladdercancer, Gallbladder Cancer, Bile Duct Cancer, PC 260 TLMDMRLSQVSCLC, PrC, CLL, OC, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer298 TMAKESSIIGV SCLC, Gallbladder Cancer, Bile Duct Cancer 110TVGGSEILFEV  SCLC, Gallbladder Cancer, Bile Duct Cancer 111 TVMDIDTSGTFSCLC, CRC, CLL, Melanoma, OC, Urinary bladder NVcancer, Gallbladder Cancer, Bile Duct Cancer, NHL 209 VFHPRQELI NSCLC261 VLFQEALWHV Urinary bladder cancer 127 VLIGSNHSLPC, BRCA, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, AML186 VLLAQIIQV Melanoma, OC, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer 262 VLPNFLPYNVNSCLC, SCLC, Brain Cancer, GC, BRCA, MCC,Melanoma, Esophageal Cancer, Urinary bladdercancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL, PC114 VLSQVYSKV SCLC 230 VLTEFTREV NSCLC, SCLC, CLL, BRCA, Melanoma, OC,Esophageal Cancer, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer, NHL 263 VLYPSLKEIRCC, BRCA, Uterine Cancer   1 VMAPFTMTI SCLC, Melanoma, NHL 264VMQDPEFLQSV SCLC, CRC, PC, CLL, Melanoma, OC, EsophagealCancer, Urinary bladder cancer, Gallbladder Cancer,Bile Duct Cancer, NHL 115 WLDDKDYFL CLL 344 VYISSLALL NSCLC, GC, CRC 265WLIEDGKVVTV Melanoma 117 YAFPKSITV PC 178 YIFTTPKSV AML 179 YIHNILYEVCLL, NHL 341 YLAIGIHEL SCLC 234 YLEPYLKEVNSCLC, SCLC, BRCA, Melanoma, OC, EsophagealCancer, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer, AML, NHL, PC  30 YLGEGPRMVNSCLC, Melanoma, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer 180 YLGPHIASVTL Melanoma121 YLITGNLEKL NSCLC, CLL, BRCA, Melanoma, OC, EsophagealCancer, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer, NHL, PC 228 YLITSVELL OC, NHL 181YLLEKFVAV NSCLC, SCLC, CLL, OC, Urinary bladder cancer, AML, NHL 269YLMEGSYNKV NSCLC, SCLC, PrC, BRCA, MCC, Melanoma, OC,Esophageal Cancer, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer 123 YLWDLDHGFANSCLC, SCLC, PrC, BRCA, Melanoma, OC GV Esophageal Cancer, AML, NHL  31YQMDIQQEL SCLC 203 YYGILQEKI NSCLC 213 YYNKVSTVF NSCLC

NSCLC=non-small cell lung cancer, SCLC=small cell lung cancer,RCC=kidney cancer, CRC=colon or rectum cancer, GC=stomach cancer,HCC=liver cancer, PC=pancreatic cancer, PrC=prostate cancer, leukemia,BRCA=breast cancer, MCC=Merkel cell carcinoma, OC=ovarian cancer,NHL=non-Hodgkin lymphoma, AML=acute myeloid leukemia, CLL=chroniclymphocytic leukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 14, 15, 41, 43, 58, 59, 60, 81, 121, 135, 139, 144,176, 236, 248, 275, 276, 283, 286, 288, 289, 290, 291, 300, 302, 304,308, 313, 316, 317, 325, 326, 329, 331, 334, 342, and 343 for the—in onepreferred embodiment combined—treatment of pancreatic cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to SEQ IDNo. 6, 15, 16, 22, 26, 30, 34, 36, 47, 59, 65, 69, 70, 77, 80, 81, 88,121, 123, 125, 127, 133, 137, 139, 169, 172, 176, 181, 186, 221, 223,229, 230, 231, 232, 233, 234, 236, 237, 238, 244, 247, 249, 250, 251,255, 260, 261, 266, 269, 271, 274, 275, 282, 285, 289, 290, 291, 293,297, 301, 302, 304, 306, 310, 313, 316, 317, 319, 327, 328, 329, 330,331, 332, 334, and 342 for the—in one preferred embodimentcombined—treatment of colon cancer or renal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to SEQ IDNo. 10, 14, 15, 22, 36, 39, 54, 55, 60, 72, 77, 81, 90, 96, 112, 116,119, 121, 133, 137, 138, 148, 169, 170, 172, 177, 186, 187, 189, 192,197, 198, 203, 206, 219, 221, 229, 230, 233, 234, 236, 255, 260, 270,272, 275, 277, 278, 279, 281, 282, 285, 289, 291, 292, 295, 296, 297,301, 302, 305, 308, 311, 313, 315, 316, 319, 321, 324, 328, 329, 333,334, 335, 336, and 346 for the—in one preferred embodimentcombined—treatment of kidney cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to SEQ IDNo. 14, 15, 16, 17, 36, 39, 47, 51, 54, 65, 88, 101, 123, 125, 133, 134,135, 137, 141, 147, 161, 166, 169, 176, 179, 184, 186, 187, 189, 191,192, 193, 194, 195, 196, 197, 199, 203, 206, 208, 214, 220, 221, 224,229, 230, 231, 234, 238, 239, 244, 245, 250, 251, 255, 258, 259, 260,268, 269, 270, 271, 272, 278, 279, 282, 295, 297, 302, 304, 305, 306,309, 310, 311, 312, 313, 316, 317, 319, 321, 325, 327, 328, 329, 330,331, 332, 333, 334, 336, 337, 338, 339, 340, 342, 343, 344, 345, and 347for the—in one preferred embodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to SEQ IDNo. 172, 173, 240, 250, 287, 299, 302, 334, and 335 for the—in onepreferred embodiment combined—treatment of CLL.

Similarly, the peptides as listed in Table 5B as above can form thebasis for the—in one preferred embodiment combined—treatment of thediseases as indicated.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofHCC, brain cancer, kidney cancer, pancreatic cancer, colon or rectalcancer, and leukemia.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 300.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofdiseases including cancer and autoimmune/inflammatory/immunepathological diseases.

The present invention further relates to antibodies against the peptidesaccording to the present invention or complexes of said peptidesaccording to the present invention with MHC, and methods of makingthese.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 300, preferably containing SEQ IDNo. 1 to SEQ ID No. 124, and SEQ ID No. 187 to SEQ ID No.: 218 or avariant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, the medicament is activeagainst cancer.

Preferably, said medicament is for a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are HCC, brain cancer, kidneycancer, pancreatic cancer, colon or rectal cancer or leukemia, andpreferably HCC cells.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of HCC. The present invention also relates to the use of thesenovel targets in the context of cancer treatment.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC molecules are composed of an alpha heavy chain andbeta-2-microglobulin (MHC class I receptors) or an alpha and a betachain (MHC class II receptors), respectively. Their three-dimensionalconformation results in a binding groove, which is used for non-covalentinteraction with peptides. MHC class I molecules can be found on mostnucleated cells. They present peptides that result from proteolyticcleavage of predominantly endogenous proteins, defective ribosomalproducts (DRIPs) and larger peptides. MHC class II molecules can befound predominantly on professional antigen presenting cells (APCs), andprimarily present peptides of exogenous or transmembrane proteins thatare taken up by APCs during endocytosis, and are subsequently processed.Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate TCR (T-cell receptor), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic S, et al. Survey of naturally occurring CD4+ T cell responsesagainst NY-ESO-1 in cancer patients: correlation with antibodyresponses. Proc Natl Acad Sci USA. 2003 Jul. 22; 100(15):8862-7). At thetumor site, T helper cells, support a cytotoxic T cell- (CTL-) friendlycytokine milieu Mortara L, et al. CIITA-induced MHC class II expressionin mammary adenocarcinoma leads to a Th1 polarization of the tumormicroenvironment, tumor rejection, and specific antitumor memory. ClinCancer Res. 2006 Jun. 1; 12(11 Pt 1):3435-43) and attract effectorcells, e.g. CTLs, NK cells, macrophages, granulocytes (Hwang M L, et al.Cognate memory CD4+ T cells generated with dendritic cell priminginfluence the expansion, trafficking, and differentiation of secondaryCD8+ T cells and enhance tumor control. J Immunol. 2007 Nov. 1;179(9):5829-38).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel J, et al.Unexpected abundance of HLA class II presented peptides in primary renalcell carcinomas. Clin Cancer Res. 2006 Jul. 15; 12(14 Pt 1):4163-70).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes. T-helper cells, activated by MHC class II epitopes,play an important role in orchestrating the effector function of CTLs inanti-tumor immunity. T-helper cell epitopes that trigger a T-helper cellresponse of the TH1 type support effector functions of CD8-positivekiller T cells, which include cytotoxic functions directed against tumorcells displaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ).

There is evidence for CD4 T cells as direct anti-tumor effectors(Braumuller et al., 2013; Tran et al., 2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was not considered possible. However,Dengjel et al. were successful in identifying a number of MHC Class IIepitopes directly from tumors (WO 2007/028574, EP 1 760 088 B1).

The antigens that are recognized by the tumor specific cytotoxic Tlymphocytes, that is, the epitopes thereof, can be molecules derivedfrom all protein classes, such as enzymes, receptors, transcriptionfactors, etc. which are expressed and, as compared to unaltered cells ofthe same origin, usually up-regulated in cells of the respective tumor.

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

The current classification of tumor associated antigens comprises thefollowing major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members or NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose; most are found inmelanomas and normal melanocytes. Many of these melanocytelineage-related proteins are involved in the biosynthesis of melanin andare therefore not tumor specific but nevertheless are widely used forcancer immunotherapy. Examples include, but are not limited to,tyrosinase and Melan-A/MART-1 for melanoma or PSA for prostate cancer.c) Overexpressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir overexpression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, Survivin, Telomerase or WT1.d) Tumor specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor-(-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

For proteins to be recognized by cytotoxic T-lymphocytes astumor-specific or -associated antigens, and to be used in a therapy,particular prerequisites must be fulfilled. The antigen should beexpressed mainly by tumor cells and not, or in comparably small amounts,by normal healthy tissues. In a preferred embodiment, the peptide shouldbe over-presented by tumor cells as compared to normal healthy tissues.It is furthermore desirable that the respective antigen is not onlypresent in a type of tumor, but also in high concentrations (i.e. copynumbers of the respective peptide per cell). Tumor-specific andtumor-associated antigens are often derived from proteins directlyinvolved in transformation of a normal cell to a tumor cell due to theirfunction, e.g. in cell cycle control or suppression of apoptosis.Additionally, downstream targets of the proteins directly causative fora transformation may be upregulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are based on the use ofT-cells that can be isolated from patients or healthy subjects, or theyare based on the generation of differential transcription profiles ordifferential peptide expression patterns between tumors and normaltissues.

However, the identification of genes over-expressed in tumor tissues orhuman tumor cell lines, or selectively expressed in such tissues or celllines, does not provide precise information as to the use of theantigens being transcribed from these genes in an immune therapy. Thisis because only an individual subpopulation of epitopes of theseantigens are suitable for such an application since a T cell with acorresponding TCR has to be present and the immunological tolerance forthis particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of TCRs and antibodies according to the invention theimmunogenicity of the underlying peptides is secondary. For TCRs andantibodies according to the invention the presentation is thedetermining factor.

Both therapeutic and diagnostic uses against additional cancerousdiseases are disclosed in the following more detailed description of theunderlying proteins (polypeptides) of the peptides according to theinvention.

Differential expression of COL18A1 was reported for bladder cancer,rhabdoid tumors and ovarian carcinoma and specific polymorphisms withinthe gene were shown to increase the risk for sporadic breast cancer(Fang et al., 2013; Gadd et al., 2010; Peters et al., 2005; Lourenco etal., 2006).

Changes in COPA gene expression and RNA editing were shown to beassociated with hepatocellular carcinoma and an experimental studyrevealed anti-apoptotic effects of COPA in mesothelioma cells (Sudo etal., 2010; Qi et al., 2014; Wong et al., 2003).

The activity of CPB2 was shown to be significantly reduced in acutepromyelocytic leukemia (Meijers et al., 2000).

CRP, an acute phase protein synthesized in the liver, was shown to be aprognostic marker in a variety of cancer types, amongst others renalcell carcinoma and multiple myeloma (Ljungberg, 2007; Fassas and Tricot,2004).

CRYZ is a target gene of the tumor-suppressor p53 (Bansal et al., 2011).Its encoded protein zeta-crystallin was shown to directly interact withthe mRNA of the anti-apoptotic molecule bcl-2 and to stabilize theover-expression of bcl-2 in T-cell acute lymphocytic leukemia (Lapucciet al., 2010).

Over-expression of CSRP2 is associated with de-differentiation ofhepatocellular carcinoma (Midorikawa et al., 2002).

CYB5A encodes an enzyme which detoxifies carcinogenic molecules and is aprognostic factor for pancreatic cancer (Blanke et al., 2014;Giovannetti et al., 2014).

Increased expression levels of CYP27A1 are associated with endometrialcarcinoma, breast cancer and colorectal cancer (Bergada et al., 2014;Nelson et al., 2013; Matusiak and Benya, 2007).

Over-expression of CYP2E1 was reported in colorectal cancer, specificpolymorphisms are associated with bladder and lung cancer and in breastcancer cells (Ye et al., 2014; Patel et al., 2014; Deng et al., 2014;Leung et al., 2013).

CYP2J2 is an enzyme, which was shown to be over-expressed in a varietyof human cancers, including esophageal, lung, breast, stomach, liver andcolon cancer (Jiang et al., 2005; Narjoz et al., 2014).

CYP4F8 was shown to be highly expressed in prostate cancer (Vainio etal., 2011). CYP4F2 and CYP4F3 were both shown to be over-expressed inpancreatic ductal adenocarcinoma and CYP4F2 alone in ovarian cancer(Gandhi et al., 2013; Alexanian et al., 2012).

Expression of CYP4F11 was shown to be regulated by NF-κB and p53(Kalsotra et al., 2004; Bell and Strobel, 2012; Goldstein et al., 2013).

Genetic variants of CYPAF12 are significantly associated withgemcitabine response in pancreatic cancer patients (Goldstein et al.,2013; Harris et al., 2014).

High levels of DAP3 correlate on the one hand with better responses tochemotherapy in gastric cancer and better clinical outcome in breastcancer, but on the other hand over-expression of DAP3 was reported inthyroid oncocytic tumors and invasive glioblastoma (Jia et al., 2014;Wazir et al., 2012; Jacques et al., 2009; Mariani et al., 2001).

PEX19 is essential for peroxisomal biogenesis, but was also shown todirectly interact with p19ARF, ultimately leading to a retention of thisfactor in the cytoplasm and to an inactivation of p53 tumor-suppressivefunction (Sugihara et al., 2001).

DDX11, belonging to the DEAH family of DNA helicases, is highlyexpressed in advanced melanoma (Bhattacharya et al., 2012).

NME4 is a nucleoside diphosphate kinase, over-expressed in colon andgastric cancer, as well as in myelodysplastic syndrome, in the latterdisease being associated with poor prognosis (Kracmarova et al., 2008;Seifert et al., 2005).

DENND5B acts as GDP-GTP exchange factor to activate Rab-GTPases(Yoshimura et al., 2010).

DIEXF was shown to mediate the non-proteasomal degradation of thetumor-suppressor p53 (Tao et al., 2013).

DOCK7 is a guanine nucleotide exchange factor, which was shown to beover-expressed in glioblastoma and to increase glioblastoma cellinvasion in response to HGF by activating Rac-1 (Murray et al., 2014).

In hepatocellular carcinoma cell lines, DRG2 was shown to bedown-regulated during chemotherapeutic drug induced apoptosis, andover-expression of DRG2 inhibits doxorubicin induced apoptosis in thesecells (Chen et al., 2012a).

DROSHA, one of the two critical enzymes in microRNA biosynthesis, isover-expressed in a number of cancers including gastrointestinal tumors,breast cancer and cervical cancer and appears to enhance proliferation,colony formation and migration of tumor cells (Avery-Kiejda et al.,2014; Havens et al., 2014; Zhou et al., 2013b).

SNPs in the DUSP14 gene are associated with altered melanoma risk (Yanget al., 2014a; Liu et al., 2013b).

A whole exome sequencing study uncovered somatic mutations within theDYNC1H1 gene in patients with intra-ductal papillary mucinous neoplasmof the pancreas (Furukawa et al., 2011).

EEF2 protein was shown to be over-expressed in lung, esophageal,pancreatic, breast and prostate cancer, in glioblastoma multiforme andin non-Hodgkin's lymphoma and to play an oncogenic role in cancer cellgrowth (Oji et al., 2014; Zhu et al., 2014a).

Mutations within the gene of EFR3A were identified in colorectal adenomasamples (Bojjireddy et al., 2014; Zhou et al., 2013a).

EIF2B5 encodes one subunit of the translation initiation factor B.Single nucleotide polymorphisms in this gene were described to beassociated with survival time in ovarian cancer (Goode et al., 2010).

EIF3A, the eukaryotic translation initiation factor 3, subunit A isover-expressed in cancers of breast, lung, cervix, esophagus, stomachand colon and was shown to be involved in cell cycle regulation (Dongand Zhang, 2006).

EIF4E is a potent oncogene elevated in up to 30% of human malignancies,including carcinomas of the breast, prostate, lung, head, and neck aswell as in many leukemias and lymphomas (Carroll and Borden, 2013).

ELOVL2 was shown to be over-expressed in hepatocellular carcinoma(Jakobsson et al., 2006; Zekri et al., 2012).

EPRS encodes a multifunctional aminoacyl-tRNA synthetase, which wasreported to be a tumor-associated antigen in colon cancer (Line et al.,2002).

EXOSC4 promoter activity is increased in hepatocellular carcinoma, dueto DNA hypomethylation. EXOSC4 effectively and specifically inhibitscancer cell growth and cell invasive capacities (Drazkowska et al.,2013; Stefanska et al., 2014).

The hydrolytic enzyme FUCA2 was found to be essential for H. pyloriadhesion to human gastric cancer cells (Liu et al., 2009a).

GABRQ encodes the GABAA receptor theta subunit. GABA was shown tostimulate human hepatocellular carcinoma growth through theover-expressed GABAA receptor theta subunit (Li et al., 2012).

In squamous cell carcinoma over-expression of GALNT2 was reported toenhance the invasive potential of tumor cells by modifyingO-glycosylation and EGFR activity (Lin et al., 2014; Hua et al., 2012a;Wu et al., 2011).

High levels of GGH have been associated with cellular resistance toanti-folates, in particular methotrexate and with poor prognosis ininvasive breast cancer and pulmonary endocrine tumors (Schneider andRyan, 2006; Shubbar et al., 2013; He et al., 2004).

GLUL is over-expressed in human breast carcinoma cells and astrocytomas(Zhuang et al., 2011; Collins et al., 1997; Christa et al., 1994;Cadoret et al., 2002).

GNPAT was reported to be implicated in growth inhibition and apoptosisinduction in metastatic melanoma (Ofman et al., 2001; Qin et al., 2013).

Deletions in the chromosomal region of GOLGA4 have been reported incervical carcinoma and in-frame mRNA fusion of GOLGA4 with PDGFRB inmyeloproliferative neoplasms (Senchenko et al., 2003; Hidalgo-Curtis etal., 2010).

GPAM is expressed in human breast cancer, which is associated withchanges in the cellular metabolism and better overall survival(Brockmoller et al., 2012).

High serum levels of GPT were reported to increase the risk ofgastrointestinal cancer and are associated with carcinogenesis andrecurrence in hepatitis C virus-induced hepatocellular carcinoma(Kunutsor et al., 2014; Tarao et al., 1997; Tarao et al., 1999).

GRB14 has been shown to be up-regulated in breast cancer, where highexpression was significantly associated with better disease-free andoverall survival (Huang et al., 2013; Balogh et al., 2012).

Single nucleotide polymorphisms in the GTF2H4 gene were reported toincrease the risk to develop smoking-related lung cancer and papillomavirus-induced cervical cancer (Mydlikova et al., 2010; Buch et al.,2012; Wang et al., 2010).

Different studies suggest an important role of HSPA2 in diseaseprogression of cervical cancer, renal cell carcinoma and bladder cancer.Polymorphisms within the gene are associated with the development ofgastric cancer (Singh and Suri, 2014; Ferrer-Ferrer et al., 2013; Garget al., 2010a; Garg et al., 2010b).

HSPA8 was shown to be over-expressed in esophageal squamous cellcarcinoma. Furthermore, HSPA8 is over-expressed in multiple myeloma andcolonic carcinoma and BCR-ABL1-induced expression of HSPA8 promotes cellsurvival in chronic myeloid leukemia (Dadkhah et al., 2013; Wang et al.,2013a; Chatterjee et al., 2013; Kubota et al., 2010; Jose-Eneriz et al.,2008).

MDN1 was described to be a candidate tumor suppressor gene, mutated inbreast cancers of the luminal B type (Cornen et al., 2014).

MIA3, also known as transport and Golgi organization protein 1 (TANGO),was reported to be down-regulated in colon and hepatocellular carcinomasand to play a tumor-suppressive role in these entities (Arndt andBosserhoff, 2007). In contrast, a study in oral squamous cell carcinomaindicates an association of MIA3 expression with tumor progression,metastasis formation and clinical stage, pointing towards an oncogenicaction of MIA3 (Sasahira et al., 2014).

CPSF6 was identified as one gene within a “poised gene cassette”associated with significant differences in metastatic and invasivepotential of several tumor types, like breast, colon, liver, lung,esophageal and thyroid cancer (Yu et al., 2008).

Low levels of MPDZ expression were reported to be associated with poorprognosis in breast cancer patients (Martin et al., 2004).

NAA35, also known as MAK10, encodes the N(alpha)-acetyltransferase 35,NatC auxiliary subunit. In patients with esophageal squamous cellcarcinoma a highly cancer enriched chimeric GOLM1-MAK10 RNA wasdetected, which encodes a secreted fusion protein, potentially useful asmolecular marker (Zhang et al., 2013b).

NAV2 was shown to be specifically expressed in a group of colon cancersand treatment of colon-cancer cells with antisense oligonucleotides forNAV2 induced apoptosis (Ishiguro et al., 2002).

NCSTN over-expression is indicative of worse overall survival inestrogen-receptor-negative breast cancer patients and high levels ofNicastrin and Notch4 were detected in endocrine therapy resistant breastcancer cells, where their activation ultimately drives invasive behavior(Sarajlic et al., 2014; Lombardo et al., 2014).

NKD1 protein is reduced, but NKD1 mRNA is elevated in non-small celllung cancer, the former correlating with increased invasive potentialand poor prognosis (Zhang et al., 2011). NKD1 mRNA was also found to beelevated in cells from human colon tumors (Yan et al., 2001; Zhang etal., 2011).

In esophageal cancer, NUDC was reported to be associated with nodalmetastasis, whereas over-expression of NUDC in prostate cancer cellsleads to a block in cell division (Hatakeyama et al., 2006; Lin et al.,2004).

A study investigating the role of the Notch signaling pathway in ovariancancer reported a higher frequency of RFNG expression in adenomacompared to carcinoma (Gu et al., 2012; Hopfer et al., 2005).

RINT1 is described as an oncogene in glioblastoma multiforme and as amoderately penetrant cancer susceptibility gene seen in breast cancer aswell as in Lynch syndrome-related cancers (Ngeow and Eng, 2014; Quayleet al., 2012).

High expression of RORC was found to be associated with longermetastasis-free survival in breast cancer. Attenuated RORC expression insomatotroph adenomas is associated with increased tumor size and ablunted clinical response to somatostatin treatment (Cadenas et al.,2014; Lekva et al., 2013).

RPL17 was reported to promote multidrug resistance by suppressingdrug-induced apoptosis (Shi et al., 2004b).

Increased expression of RPS29 was reported in gastric and colorectalcancer (Takemasa et al., 2012; Sun et al., 2005).

SAMM50 encodes a component of the Sorting and Assembly Machinery (SAM)of the mitochondrial outer membrane, which functions in the assembly ofbeta-barrel proteins into the outer mitochondrial membrane. A growthpromoting chimeric mRNA (SAMM50-PARVB) was detected in breast andovarian cancer cells and in a number of samples from breast, stomach,colon, kidney and uterus cancer (Plebani et al., 2012).

SERPINF2 encodes the major inhibitor of plasmin, which degrades fibrinand various other proteins. The plasma level of the plasmin-alpha2-plasmin inhibitor complex was shown to be a predictor of survival innon-small cell lung carcinoma and low activity of alpha 2-antiplasminhas been observed in the blood of the patients with prostatic carcinoma(Zietek et al., 1996; Taguchi et al., 1996).

Over-expression of SF3B3 is significantly correlated with overallsurvival and endocrine resistance in estrogen receptor-positive breastcancer (Gokmen-Polar et al., 2014).

Protein levels of SHC1 are elevated in prostate, metastatic breast,ovarian and thyroid cancer and different isoforms and are thought tofunction as a primary adaptor protein for mediating the mitogenicsignals of steroids at the non-genomic level (Alam et al., 2009;Rajendran et al., 2010).

AMACR is used as a biomarker in prostate cancer, since it is highlyover-expressed in this entity (Wu et al., 2014). Furthermore, it is usedas an immunohistochemical marker for the diagnosis of renal cellcarcinoma (Ross et al., 2012).

Experimental data suggest that C1QTNF3 expression may play a role inosteosarcoma tumor growth, associated with activation of the ERK1/2signaling pathway and that it is a novel anti-apoptotic adipokine thatprotects mesenchymal stem cells from hypoxia/serum deprivation-inducedapoptosis through the PI3K/Akt signaling pathway (Hou et al., 2014;Akiyama et al., 2009).

GPC3 is expressed by most hepatocellular carcinomas. Two therapeuticapproaches for HCC that target GPC3 are currently being tested in phaseII clinical trials: a humanized GPC3 monoclonal antibody and a vaccinethat consists of two GPC3-derived peptides. The peptides used in thelatter study are distinct from the peptide presented in this document.GPC3 expression has also been identified in all yolk sac tumors, somesquamous cell carcinomas of the lung and clear cell carcinomas of theovary (Filmus and Capurro, 2013; Kandil and Cooper, 2009).

MAGEB2 is classified as cancer testis antigen, since it is expressed intestis and placenta, and in a significant fraction of tumors of varioushistological types, amongst others multiple myeloma and head and necksquamous cell carcinoma (Pattani et al., 2012; van et al., 2011).

MAPKAPK5 encodes a tumor suppressor and member of the serine/threoninekinase family. MAPKAPK5 was shown to be under-expressed in colorectalcancer, leading to an increased activity of the myc oncoprotein and todecrease cancer formation by suppressing oncogenic ras activity in amurine model of hematopoietic cancer (Yoshizuka et al., 2012; Kress etal., 2011).

Over-expression of USP14 is associated with increased tumor cellproliferation and poor prognosis in epithelial ovarian, non-small celllung and colorectal cancer (Wang et al., 2015; Wu et al., 2013a; Shinjiet al., 2006).

C4A has been described as a biomarker for polycystic ovary syndrome andendometrial cancer and experimental data suggest that C4 can mediatecancer growth (Galazis et al., 2013; Rutkowski et al., 2010).

CAPZB was reported to be over-expressed in human papillomaviruses18-positive oral squamous cell carcinomas and was identified as prostatecancer susceptibility locus (Lo et al., 2007; Nwosu et al., 2001).

Single nucleotide polymorphisms within the gene for CFHR5 are associatedwith event-free survival in follicular lymphoma (Charbonneau et al.,2012).

CLIP1 encodes the CAP-GLY domain containing linker protein 1, whichlinks endocytic vesicles to microtubules. This gene is highly expressedin Reed-Sternberg cells of Hodgkin disease and breast cancer and appearsto be implicated in the migration and invasion of breast cancer andpancreatic cancer cells (Sun et al., 2013; Suzuki and Takahashi, 2008;Li et al., 2014a; Sun et al., 2012).

CLU may inhibit tumor progression, whereas in advanced neoplasia, it mayoffer a significant survival advantage in the tumor by suppressing manytherapeutic stressors and enhancing metastasis. CLU has been shown toplay a critical role in prostate cancer pathogenesis, to regulate theaggressive behavior of human clear renal cell carcinoma cells throughmodulating ERK1/2 signaling and MMP-9 expression and to conferresistance to treatment in advanced stages of lung cancer (Trougakos,2013; Panico et al., 2009; Takeuchi et al., 2014; Wang et al., 2014).

The fusion gene SEC16A-NOTCH1 was reported as first recurrent fusiongene in breast cancer (Edwards and Howarth, 2012).

Recurrent deletion of the SHQ1 gene has been observed in prostate andcervical cancer, implicating a tumor-suppressive role of SHQ1 (Krohn etal., 2013; Lando et al., 2013).

In clear cell renal cell carcinomas and bladder cancer high SLC16A1expression is associated with poor prognostic factors and predicts tumorprogression. In colorectal cancer single nucleotide polymorphisms in theSLC16A1 gene may affect clinical outcomes and can be used to predict theresponse to adjuvant chemotherapy (Kim et al., 2015; Fei et al., 2014a;Fei et al., 2014a).

Glioblastoma have been shown to release glutamate at high levels, whichmay stimulate tumor cell proliferation and facilitates tumor invasion,and to down-regulate SLC1A2, which correlated with higher tumor grade,implicating its potential role in glial tumor progression. Furthermore,in gastric cancer a fusion gene of SLC1A2 with CD44 has been detectedand may represent a class of gene fusions that establish a pro-oncogenicmetabolic milieu favoring tumor growth and survival (Tao et al., 2011;de Groot et al., 2005).

High expression of SLC3A2 is associated with tumor growth, biologicalaggressiveness, and survival of patients with biliary tract cancer andsignificantly contributes to poor prognosis of non-small cell lungcancer patients through promoting cell proliferation via the PI3K/Aktpathway. Furthermore, over-expression of SLC3A2 together with integrinβ1, integrin β3 and Fak is associated with the progression and livermetastases of colorectal cancer (Kaira et al., 2014; Fei et al., 2014b;Sun et al., 2014).

Evidences of SLC9A3R1 involvement in cancer development are present inhepatocellular carcinoma, schwannoma, glioblastoma, colorectal cancerand particularly in breast cancer (Saponaro et al., 2014).

NFYC has been reported to promote the expression of oncogenes in gastriccancer and prostate cancer cells (Zhang et al., 2014a; Gong et al.,2013).

THY1 is a candidate tumor suppressor gene in nasopharyngeal carcinomabearing anti-invasive activity (Lung et al., 2010).

TIMM17A is over-expressed in 21T breast cancer cells and mRNA expressionin breast cancer tissues was correlated with tumor progression (Xu etal., 2010).

TMEM209 is widely expressed in lung cancer (Fujitomo et al., 2012).

TNK2 also known as ACK1 tyrosine kinase is activated, amplified ormutated in a wide variety of human cancers. The de-regulated kinase isoncogenic and its activation correlates with progression to metastaticstage. ACK1 inhibitors have shown promise in pre-clinical studies(Mahajan and Mahajan, 2013).

TRIM55 encodes a RING zinc finger protein which associates transientlywith microtubules, myosin and titin during muscle sarcomere assembly andis also involved in signaling from the sarcomere to the nucleus (Pizonet al., 2002).

RNA interference of Ufd1 protein can sensitize ahydroxycamptothecin-resistant colon cancer cell line SW1116/HCPT tohydroxyl-camptothecin (Chen et al., 2011a; Chen et al., 2011c).

In colorectal cancer the UGT1A1 gene is silenced through methylation andthus is regarded as the target point of research for irinotecan (CPT-11)drug resistance and control mechanisms for the reversal of drugresistance (Xie et al., 2014).

UGT1A10 is expressed in gastric and biliary tissue (Strassburg et al.,1997) and its over-expression significantly increased the cytotoxicityof the antitumor agent5-dimethylaminopropylamino-8-hydroxytriazoloacridinone C-1305 (Pawlowskaet al., 2013). Furthermore UGT1A10 catalyzes the glucuronidation ofxenobiotics, mutagens, and reactive metabolites and thus acts asindirect antioxidant. Xenobiotic (XRE) and antioxidant (ARE) responseelements were detected in the promoters of UGT1A8, UGT1A9, and UGT1A10(Kalthoff et al., 2010).

UGT1A8 is primarily expressed in the gastrointestinal tract (Gregory etal., 2003) and mRNA expression is up-regulated upon treatment withchemo-preventive agent sulforaphane (SFN) (Wang et al., 2012).

UGT1A7 haplotype is associated with an increased risk of hepatocellularcarcinoma in hepatitis B carriers (Kong et al., 2008).

UGT1A6 is over-expressed in breast cancer cells resistant tomethotrexate (de Almagro et al., 2011) and induced by β-Naphthoflavone aputative chemo-preventive agent (Hanioka et al., 2012).

UGT1A9 is mainly expressed in liver and kidneys (Gregory et al., 2003).UGT1A9 germline polymorphisms are potential predictors for prostatecancer recurrence after prostatectomy (Laverdiere et al., 2014).

UGT1A4 promoter and coding region polymorphisms lead to a variability inthe glucuronidation of anastrozole, an aromatase inhibitor for breastcancer patients (Edavana et al., 2013).

UPF1 is part of the nonsense-mediated mRNA decay (NMD) machinery and mayhave a functional role in prostate cancer progression and metastasis(Yang et al., 2013). Further the UPF1 RNA surveillance gene is commonlymutated in pancreatic adenosquamous carcinoma (Liu et al., 2014).

UQCRB is a subunit of mitochondrial complex III. Inhibition of UQCRB intumor cells suppresses hypoxia-induced tumor angiogenesis (Jung et al.,2013). Two SNPs in the 3′ untranslated region of UQCRB are candidates asprognostic markers for colorectal cancer (Lascorz et al., 2012).

Copy number alterations of USO1 correlated with differential geneexpression in superficial spreading melanoma compared to nodularmelanoma (Rose et al., 2011).

Significant reductions in both USP10 and SIRT6 protein expression wasdetected in human colon cancers (Lin et al., 2013).

UTP18 also alters translation to promote stress resistance and growth,and is frequently gained and over-expressed in cancer (Yang et al.,2014b).

VARS rs2074511 polymorphism was associated with survival in patientswith triple negative type breast cancer and thus may be considered as aprognostic factor for survival in patients with early breast cancer(Chae et al., 2011).

VMP1, a stress-induced autophagy-associated protein, is also induced bythe oncogene KRAS (Lo Re et al., 2012). VMP1 is over-expressed in poorlydifferentiated human pancreatic cancer as a response to chemotherapeuticdrugs (Gilabert et al., 2013). A significant down-regulation of VMP1 wasfound in human HCC tissues and closely correlated with multiple tumornodes, absence of capsular formation, vein invasion and poor prognosisof HCC (Guo et al., 2012).

WDR26 protects myocardial cells against oxidative stress (Feng et al.,2012).

ZC3H7A is part of the CCCH zinc finger protein family known asregulators of macrophage activation (Liang et al., 2008). ZC3H7A wasfound to have higher allele frequencies of functional mutations in themetastatic tumor of pancreatic ductal adenocarcinoma (Zhou et al.,2012).

FASN is a fatty acid synthase and involved in the enhanced lipidsynthesis in different types of cancer, including breast, pancreatic,prostate, liver, ovarian, colon and endometrial cancer (Wu et al., 2014;Zhao et al., 2013).

FGG is up-regulated in hepatocellular carcinoma as well as in prostate,lung and breast cancers (Vejda et al., 2002; Zhu et al., 2009).

FMO5 is a monooxygenase that is the dominant liver-specific FMO, and itis up-regulated in estrogen receptor alpha-positive breast tumors(Bieche et al., 2004; Zhang and Cashman, 2006).

HADHA mRNA is reduced with the progression of de-differentiation in HCC(Tanaka et al., 2013) and in estrogen receptor alpha-negative breasttumors (Mamtani and Kulkarni, 2012).

Genetic variation in the HAL gene might play a role in the developmentof skin cancer (Welsh et al., 2008).

HLTF is a member of the SWI/SNF family of transcriptional regulatorswith helicase and E3 ubiquitin ligase activity and was found to beinactivated by hypermethylation in colon, gastric, uterine, bladder andlung tumors (Debauve et al., 2008; Castro et al., 2010; Garcia-Baqueroet al., 2014).

HDAC10 is a histone deacetylase and transcriptional regulator.Expression of HDAC10 was significantly decreased in gastric cancertissues as compared with adjacent tissues (Jin et al., 2014). HDAC10 isinversely related to lymph node metastasis in human patients withcervical squamous cell carcinoma (Song et al., 2013). HDAC10 ishypermethylated in malignant adrenocortical tumors (Fonseca et al.,2012). HDAC10 levels are increased in chronic lymphocytic leukemia (Wanget al., 2011). HDAC10-589C>T promoter polymorphism was significantlyassociated with HCC occurrence among chronic HBV patients as well as HCCacceleration among chronic HBV patients (Park et al., 2007). Reducedexpression of class II histone deacetylase genes is associated with poorprognosis in lung cancer patients (Osada et al., 2004).

Low HIP1R expression is strongly associated with poor outcome in diffuselarge B-cell lymphoma patients (Wong et al., 2014).

HM13 is a signal peptide peptidase and affected cell viability incolorectal adenoma (Sillars-Hardebol et al., 2012).

Serum HPR levels in patients with malignant lymphoma were significantlyhigher than in non-diseased control groups and HPR expression increasedwith disease progress (Epelbaum et al., 1998). HPR expression parallelsincreased malignant potential in breast cancers and HPR-positive breastcancers are more likely to recur after primary resection and areassociated with shorter disease-free intervals (Shurbaji et al., 1991).

A variant (rs932335) in the HSD11B1 gene is associated with colorectalcancer and breast cancer (Feigelson et al., 2008; Wang et al., 2013b).

HSD17B6 expression in tissues from prostate cancer patients undergoingandrogen deprivation therapy (ADT) was significantly higher than that intissues of untreated individuals (Ishizaki et al., 2013).

HSPE1 is a mitochondrial chaperonin with functions in protein foldingand cell signaling (NF-kappaB and WNT signaling). Increased Hsp10 levelshave been found in tumor cells in large bowel cancer, exocervicalcancer, prostate cancer, mantle cell lymphoma, and serous ovariancancer. In bronchial carcinogenesis, decreased levels of Hsp10 have beenreported (David et al., 2013).

Ovarian carcinoma xenografts transplanted into the flanks of nude miceand treated with paclitaxel showed a diminished IDI1 expression comparedto untreated xenograft (Bani et al., 2004).

IGFBPL1 is a regulator of insulin-growth factors and is down-regulatedin breast cancer cell lines by aberrant hypermethylation. Methylation inIGFBPL1 was clearly associated with worse overall survival anddisease-free survival (Smith et al., 2007).

The androgen-sensitive microsome-associated protein IKBKAP modulated theexpression of prostate epithelial and neuronal markers, attenuatedproliferation through an androgen receptor-dependent mechanism, andco-regulated androgen receptor-mediated transcription in LNCaP prostateadenocarcinoma cells (Martinez et al., 2011).

INTS8 is part of a marker panel that discriminates gastric carcinomasfrom adjacent noncancerous tissues (Cheng et al., 2013).

A IRS2-derived peptide pIRS-21097-1105 was presented on HLA-A2(+)melanomas and breast, ovarian, and colorectal carcinomas (Zarling etal., 2014). IRS-2 1057 DD genotype and D allele were significantlyassociated with HCC risk (Rashad et al., 2014).

ITGA7 is the alpha chain of the laminin-1 receptor dimer integrinalpha-7/beta-1. ITGA7 is a tumor-suppressor gene that is critical forsuppressing the growth of malignant tumors. Mutational analysis revealedITGA7 mutations in prostate cancer, hepatocellular carcinoma, softtissue leiomyosarcoma, and glioblastoma multiforme. ITGA7 wasdown-regulated in non-metastatic prostate cancer and leiomyosarcoma (Tanet al., 2013).

ITIH4 was down-regulated in several tumor tissues including colon,stomach, ovary, lung, kidney, rectum and prostate (Hamm et al., 2008).Low serum ITIH4 levels are associated with shorter survival inHBV-associated HCC patients (Noh et al., 2014).

Significantly increased ITIH4 serum concentrations were observed inbreast cancer and ITIH4 serum levels were significantly decreased aftersurgery (van, I et al., 2010).

A missense mutation was identified in SHKBP1, which acts downstream ofFLT3, a receptor tyrosine kinase mutated in about 30% of AML cases(Greif et al., 2011). SHKBP1 is one of several potential proteinbiomarker candidates for classifying well-differentiated small intestineneuroendocrine tumors (WD-SI-NETs) at different stage of disease(Darmanis et al., 2013).

KLB expression is elevated in HCC tissues compared to matched non-tumortissue (Poh et al., 2012).

The LBP polymorphism rs2232596 is associated with a significantlyincreased risk of colorectal carcinoma in Han Chinese (Chen et al.,2011b). LBP is a candidate serum biomarker in ovarian carcinoma (Boylanet al., 2010). LBP was reduced significantly after treatment withchemotherapy in small-cell lung carcinoma patients (Staal-van den BrekelA J et al., 1997).

LBR mRNA expression was directly associated with tumor grade andNottingham Prognostic Index in breast cancer (Wazir et al., 2013). LBRis heavily expressed in papillary thyroid carcinoma cells, but anabnormal folding of the protein might explain its lack ofimmunohistochemical reactivity and be associated with an anomalousfolding of the nuclear membrane (Recupero et al., 2010).

LEPR dysregulation has been reported in a variety of malignant cellsincluding colon cancer, hepatocellular carcinoma, endometrial cancer,thyroid cancer, breast cancer and lung cancer (Ntikoudi et al., 2014;Surmacz, 2013; Uddin et al., 2011).

LIG1 single-nucleotide polymorphisms are associated with the risk oflung cancer, endometrial cancer and glioma (Doherty et al., 2011; Lee etal., 2008; Liu et al., 2009b).

LRPPRC expression in gastric cancer tissues is significantly higher thanthat in paired control tissue (Li et al., 2014b). LRPPRC levels serve asa prognosis marker of patients with prostate adenocarcinomas (PCA), andpatients with high LRPPRC levels survive a shorter period after surgerythan those with low levels of LRPPRC (Jiang et al., 2014). LRPPRC isabundantly expressed in various types of tumors, such as lungadenocarcinoma, esophageal squamous cell carcinoma, stomach, colon,mammary and endometrial adenocarcinoma, and lymphoma (Tian et al.,2012).

MANEA expression is regulated by androgens in prostate cancer cells(Romanuik et al., 2009).

OPLAH is expressed in lung, breast, kidney, colon and ovary normal andtumor tissues and OPLAH levels are significantly higher in normalspecimens than tumors for individual patients (Srivenugopal andAli-Osman, 1997).

ORM2 glycoforms provide valuable information for differentiation betweenprimary and secondary liver cancer (Mackiewicz and Mackiewicz, 1995).ORM2 levels in plasma were confirmed to be significantly elevated inpatients suffering from colorectal carcinoma compared with the controls(Zhang et al., 2012). Fucosylated glycoform ORM2 levels weresignificantly higher in adenocarcinoma lung cancer cases compared tocontrols (Ahn et al., 2014). ORM2 is a putative biomarkers for earlydiagnosis of cholangiocarcinoma (Rucksaken et al., 2012).

Increased tetrahydrobiopterin levels result in an enhancement of PAHactivity and PAH protein in human hepatoma cells (McGuire, 1991).

PARP14 is highly expressed in myeloma plasma cells and associated withdisease progression and poor survival. PARP14 is critically involved inJNK2-dependent survival. PARP14 was found to promote the survival ofmyeloma cells by binding and inhibiting JNK1 (Barbarulo et al., 2013).

PC levels are elevated in liver tumors and lung cancer (Chang andMorris, 1973; Fan et al., 2009).

Increased PCNT levels and centrosomal abnormalities have been describedin a variety of hematologic malignancies and solid tumors, includingAML, CML, mantle cell lymphoma, breast cancer and prostate cancer(Delaval and Doxsey, 2010).

PIGN is a cancer chromosomal instability (CIN)-suppressor gene that issubject to frequent copy number loss in CIN(+) colorectal cancer(Burrell et al., 2013).

PIPOX expression varied according to subtype of breast cancer, withHER-2 type tumors showing elevated expression and triple negative breastcancer subtype showing decreased expression. Tumoral PIPOX negativitywas associated with shorter disease-free survival (Yoon et al., 2014).PIPOX was reduced in prostate tumors and reduced the oncogenic potentialof prostate cells by metabolizing sarcosine (Khan et al., 2013).

Increased PSMD4 levels were detected in colon cancer, myeloma andhepatocellular carcinoma (Arlt et al., 2009; Midorikawa et al., 2002;Shaughnessy, Jr. et al., 2011).

PLIN2 is significantly increased in patients with clear cell andpapillary renal cell carcinoma compared with controls. The preoperativeurinary concentrations of PLIN2 reflects the tumor size and stage(Morrissey et al., 2014). PLIN2 expression is significantly higher inlung adenocarcinoma specimens than in normal tissues and lung squamouscell carcinomas (Zhang et al., 2014b).

PLK4 frequently undergoes rearrangement or loss in human cancers, at aparticularly high rate in hepatocellular carcinomas, but also incolorectal cancer, head and neck cancer (Swallow et al., 2005). PLK4 isover-expressed in breast cancer (Marina and Saavedra, 2014).

QARS is a member of the aminoacyl-tRNA synthetases (ARS) and chargestRNAs with glutamine. ARS expression and polymorphisms are associatedwith breast cancer and glioblastoma (He et al., 2014b; Kim et al.,2012).

The methylated PMF1 gene is a diagnostic and prognostic biomarker forpatients with bladder cancer (Kandimalla et al., 2013).

Several human tumors and hematologic malignancies up-regulated PON2,including thyroid gland, prostate, pancreas, testis, endometrium/uterus,liver and kidney cancer, lymphoid tissues, urinary bladder tumors, ALLand CML, and such over-expression provided resistance to differentchemotherapeutics (imatinib, doxorubicine, staurosporine, oractinomycin) (Witte et al., 2011).

PRKAR2A is a regulatory subunit of protein kinase A. PRKAR2A markedlyincreased survival of prostate cancer cells lines treated with Taxol andTaxotere (Zynda et al., 2014). PRKAR2A is over-expressed in lungadenocarcinoma (Bidkhori et al., 2013).

PRPF6 is a member of the tri-snRNP (small ribonucleoprotein) spliceosomecomplex that drives colon cancer proliferation by preferential splicingof genes associated with growth regulation (Adler et al., 2014). PRPF6is over-expressed in lung adenocarcinoma (Bidkhori et al., 2013).

PSMC4 is significantly and coherently up-regulated in prostate carcinomacells compared with the corresponding adjacent normal prostate tissue(Hellwinkel et al., 2011).

QPRT expression increases with malignancy in glioma and, in recurrentglioblastomas after radiochemotherapy, QPRT expression is associatedwith a poor prognosis (Sahm et al., 2013). QPRT is a potential markerfor the immunohistochemical screening of follicular thyroid nodules(Hinsch et al., 2009).

RABGGTB is over-expressed in chemotherapy-refractory diffuse largeB-cell lymphoma (Linderoth et al., 2008).

RAD21 is over-expressed in gastrointestinal tumors, colorectalcarcinoma, advanced endometrial cancer, prostate cancer and breastcancer (Atienza et al., 2005; Deb et al., 2014; Porkka et al., 2004;Supernat et al., 2012; Xu et al., 2014).

RAD23B has a potential role in breast cancer progression (Linge et al.,2014). The single nucleotide polymorphism RAD23B rs1805329 wassignificantly associated with development and recurrence of HCC inJapanese patients with HCV (Tomoda et al., 2012).

RASAL2 is a RAS-GTPase-activating protein with tumor suppressorfunctions in estrogen receptor-positive breast cancer, ovarian cancerand lung cancer (Li and Li, 2014; Huang et al., 2014). In contrast,RASAL2 is oncogenic in triple-negative breast cancer and drivesmesenchymal invasion and metastasis (Feng et al., 2014a).

Depletion of RNMT effectively and specifically inhibits cancer cellgrowth and cell invasive capacities in different types of cancer,inclusing liver cancer (Stefanska et al., 2014).

Over-expression of ROCK1 or mutations in the ROCK1 gene that lead to anelevated kinase activity have been reported for several cancers,including lung cancer, gastric carcinoma, CML and AML (Rath and Olson,2012).

RPL10A is a c-Myc targeted gene and may contribute to hepatocytetransformation (Hunecke et al., 2012).

Inv(3) and t(3; 3) breakpoints, which are associated with a particularlypoor prognosis in myeloid leukemia or myelodysplasia, cluster in aregion that is located centromeric and downstream of the RPN1 gene(Wieser, 2002).

RRBP1 is over-expressed in lung cancer and breast cancer (Telikicherlaet al., 2012; Tsai et al., 2013).

SCFD1 expression is increased in erosive gastritis, which is linked togastric carcinoma (Galamb et al., 2008).

ABCB1 encodes P-glycoprotein (P-gp) which is expressed in normal cellsof various organs such as intestine, liver, kidney, brain, and placenta.P-gp overexpression and genetic polymorphisms have been detected incolorectal carcinoma, tumors derived from the adrenal gland, lung cancerand ALL (Zhang et al., 2013a; Fojo et al., 1987; Gervasini et al., 2006;Jamroziak et al., 2004).

ABCB10 encodes for an ABC transporter of the sub-family B (MDR/TAP).ABCB10 was shown to be involved in the cisplatin resistance of KCP-4human epidermoid carcinoma cells (Oiso et al., 2014).

The expression of ABCB11 was shown to be up-regulated in the pancreaticductal adenocarcinoma, one of the most drug-resistant cancers. Thus itmay contribute to the generally poor treatment response of this cancer(Mohelnikova-Duchonova et al., 2013).

The up-regulated expression of ABCC2 in primary fallopian tubecarcinomas is associated with poor prognosis (Halon et al., 2013).

ABCC6 was down-regulated in colorectal cancer of non-responders topalliative chemotherapy (Hlavata et al., 2012). In contrast, it wasup-regulated in the gemcitabine-resistant human NSCLC A549 cells (Ikedaet al., 2011).

The expression of ACACA was shown to be up-regulated in numerous humancancers, such as breast, prostate and liver carcinoma and correlatedwith enhanced lipogenesis of cancer cells. The various ACACA inhibitorsshowed a therapeutical effect in treatment of cancer cell lines bysuppression of cell proliferation and inducing of cell death throughapoptosis (Zu et al., 2013).

ACLY is aberrantly expressed in various tumors, such as breast, liver,colon, lung and prostate cancers, and is correlated reversely with tumorstage and differentiation (Zu et al., 2012).

ACSL3 is over-expressed in lung cancer and based on preclinicalinvestigation is a promising new therapeutic target in lung cancer (Peiet al., 2013). The up-regulated expression of ACSL3 can serve as apotential biomarker of estrogen receptor-specific breast cancer risk(Wang et al., 2013c).

ACSL4 is over-expressed in estrogen receptor-negative breast tumors andandrogen receptor-negative breast and prostate tumors. The loss ofsteroid hormone sensitivity was associated with induction of ACSL4expression (Monaco et al., 2010). The onset up-regulation of ACSL4 wasshown to occur during the transformation from adenoma to adenocarcinoma(Cao et al., 2001).

The methylation of ACSS3 was found to be associated with at least one ofthe classical risk factors, namely age, stage or MYCN status inneuroblastoma (Decock et al., 2012).

The deletion of ADSSL1 was frequently observed in carcinogen-inducedmouse primary lung adenocarcinomas, mouse and human lung adenocarcinomascell lines and associated with a more extensive chromosome instabilityphenotype in the primary mouse lung tumors (Miller et al., 2009).

AGFG2 was identified to be one of 14 prognostic gene candidates inidentifying cases of hormone receptor-negative or triple-negative breastcancers likely to remain free of metastatic relapse (Yau et al., 2010).

AGT is a very potent anti-angiogenic factor, which was shown to exertanti-tumoral effects in vitro and in vivo (Bouquet et al., 2006). Intransgenic mice, the over-expression of human AGT was shown to decreaseangiogenesis and thus delaying tumor progression of hepatocarcinoma(Vincent et al., 2009).

AKR1C4 encodes for a human aldo-keto reductase family 1 member C4 andcatalyzes the reduction of retinaldehyde to retinol (Ruiz et al., 2011).Thus, the depletion of retinaldehyde down-regulates the biosynthesis ofretinoic acid and is followed by blockage of retinoid signaling, whichfavors tumor progression (Tang and Gudas, 2011; Ruiz et al., 2012)

The expression of ALDH1L1 was shown to be down-regulated in HCC andgliomas. The down-regulation of ALDH1L1 in those cancers was associatedwith poorer prognosis and more aggressive phenotype (Rodriguez et al.,2008; Chen et al., 2012b)

The expression of ALG3 was shown to be enhanced in esophageal squamouscell carcinoma and cervical cancer (Shi et al., 2014; Choi et al.,2007). In esophageal squamous cell carcinoma the increased expression ofALG3 correlated with lymph node metastasis (Shi et al., 2014).

ANKS1A was identified as a novel target of Src family kinases which areknown to be implicated in the development of some colorectal cancers(Emaduddin et al., 2008).

APOA1 encodes for apolipoprotein A-I, the major protein component ofhigh density lipoprotein (HDL) in plasma. In multiple animal tumormodels, APOA1 showed a potent immune-modulatory role in thetumorigenesis and was shown to suppress tumor growth and metastasis bysupporting innate and adaptive immune processes (Zamanian-Daryoush etal., 2013).

APOA2 was shown to be significantly decreased in pancreatic cancerpatients (Honda et al., 2012). In contrast, the increased expression ofAPOA2 was associated with HCC (Liu et al., 2007).

In alpha-fetoprotein-negative HBV-related HCC, APOB was found to be oneof the 14 differentially expressed proteins which could be associatedwith HCC progression (He et al., 2014a). In advanced breast cancer, APOBwas found to be the one of six differentially expressed proteins whichcould predict the responsiveness to neoadjuvant chemotherapy andrelapse-free survival of patients (Hyung et al., 2011).

By stage III colorectal cancer patients and in human melanoma cells,AQP9 was associated with increased chemoresistance (Dou et al., 2013;Gao et al., 2012).

ARG1 was shown to be a sensitive and specific marker in distinguishingof HCC from other metastatic tumors in liver (Sang et al., 2013). ARG1may contribute to local immune suppression in NSCLC (Rotondo et al.,2009).

The phosphorylated and thus more active form of ARSB protein was foundto be increased in peripheral leukocytes from patients with chronicmyelogenous leukemia compared to healthy donors (Uehara et al., 1983).

In ovarian cancer cells, the down-regulation of ASNA1 was shown toincrease the sensitivity to the chemotherapy drugs cisplatin,carboplatin, oxaliplatin and arsenite (Hemmingsson et al., 2009).

ASPH was shown to be over-expressed in various cancers and cancer celllines (Yang et al., 2010). Immunization with ASPH-loaded dendritic cellsgenerated cytotoxicity against cholangiocarcinoma cells in vitro andsignificantly suppressed intrahepatic tumor growth and metastasis (Nodaet al., 2012).

ATP1A2 was found among 31 proteins which were significantly up-regulatedin glioblastoma (Com et al., 2012). In contrast, ATP1A2 was shown to bedown-regulated in bone marrow-infiltrating metastatic neuroblastomas(Morandi et al., 2012).

ATP1A3 was found among 31 proteins, which were significantlyup-regulated in glioblastoma (Com et al., 2012).

ATP6V1C1 may promote breast cancer growth and bone metastasis throughregulation of lysosomal V-ATPase activity. ATP6V1C1 knockdownsignificantly inhibited mouse 4T1 mammary tumor cell xenograft tumorgrowth, metastasis, and osteolytic lesions in vivo (Feng et al., 2013).ATP6V1C1 was shown to be over-expressed in oral squamous cell carcinomaand was associated with tumor cell mobility (Otero-Rey et al., 2008).

ATP7B is associated with cancer resistance to cisplatin, the widely usedanti-cancer drug (Dmitriev, 2011).

AXIN2 encodes for Axin- (axis inhibition) related protein 2, whichpresumably plays an important role in the regulation of the stability ofbeta-catenin in the Wnt signaling pathway (Salahshor and Woodgett,2005). Furthermore, AXIN2 was shown to repress the expression of theoncogene c-MYC (Rennoll et al., 2014).

In HCC, the low expression of BAAT was associated with poorer survivalcompared to the patients with higher expression of BAAT (Furutani etal., 1996).

A strong decrease of transcripts of BHMT and BHMT2 was shown in HepG2cells and in HCC samples compared to normal liver tissue (Pellanda etal., 2012).

C12orf44 was shown to be essential for autophagy and interact with ULK1in an Atg13-dependent manner (Mercer et al., 2009). Autophagy has dualroles in cancer, acting as both a tumor suppressor by preventing theaccumulation of damaged proteins and organelles and as a mechanism ofcell survival that can promote the growth of established tumors (Yang etal., 2011 b).

C17orf70 is a component of the Fanconi anemia core complex and isessential for the complex stability. The Fanconi anemia core complexplays a central role in the DNA damage response network. The Fanconianemia core complex-mediated DNA damage response involves breast cancersusceptibility gene products, BRCA1 and BRCA2 (Ling et al., 2007).

C19orf80 encodes for hepatocellular carcinoma-associated gene TD26 andwas shown to be one of 5 loci with highest methylation levels in HCC andlowest in control tissue (Ammerpohl et al., 2012).

CCT7 was found to be a part of a protein sub-network, which issignificantly discriminative of late stage human colorectal cancer(Nibbe et al., 2009).

CDK6 has been shown to regulate the activity of tumor suppressor proteinRb. CDK6 can exert its tumor-promoting function by enhancingproliferation and stimulating angiogenesis (Kollmann et al., 2013). Thepharmacological inhibition of CDK6 was shown to inhibit the growthdifferentiation of abnormal leukemic cells (Placke et al., 2014).

CFH may play a role in cutaneous squamous cell carcinoma progression(Riihila et al., 2014). CFH may play a key role in the resistance ofcomplement-mediated lysis in various cancer cells and was shown to beover-expressed in NSCLC, which was associated with poorer prognosis (Cuiet al., 2011).

An inactivating mutation of CLPTM1 was found in prostate cancer cells(Rossi et al., 2005).

CMAS encodes for cytidine monophosphate N-acetylneuraminic acidsynthetase, which catalyzes the activation of sialic acid and itstransformation to a cytidine monophosphate diester. The activated sialicacid is used for N-glycosylation, a common post-translationalmodification during cellular differentiation. The increased expressionof sialic acid sugars on the surface of cancer cells is one ofwell-known tumor characteristics (Bull et al., 2014).

TF (Transferrin) is one of the most widely used tumor-targeted ligands,because TF receptors (TFRs) are over-expressed on malignant cells andplay a key role in cellular iron uptake through the interaction with TF(Biswas et al., 2013). The expression level of TFRs has been suggestedto correlate with tumor stage or cancer progression (Tortorella andKaragiannis, 2014).

TH1L might play an important role in regulation of proliferation andinvasion in human breast cancer, and could be a potential target forhuman breast cancer treatment (Zou et al., 2010).

THTPA hydrolysis might be responsible for the anti-proliferative effectsof Ndrg-1. Ndrg-1 has been shown to reduce the invasion and metastasisof breast, colon, prostate and pancreatic cancer (Kovacevic et al.,2008).

SMYD3 promotes cancer invasion by epigenetic up-regulation of themetalloproteinase MMP-9 (Medjkane et al., 2012). Expression of SMYD3 isundetectable or very weak in many types of normal human tissue, whereasover-expression of SMYD3 has been linked with the development andprogression of gastric, colorectal, hepatocellular, prostate and breastcancers (Hamamoto et al., 2006; Liu et al., 2014; Liu et al., 2013a).

A link between STAT2 and tumorigenesis was observed in transgenic micelacking STAT2 (Yue et al., 2015) or expressing constitutively IFN-α inthe brain (Wang et al., 2003).

TACC3 is over-expressed in many human cancers, including ovarian cancer,breast cancer, squamous cell carcinoma and lymphoma (Ma et al., 2003;Jacquemier et al., 2005; Lauffart et al., 2005).

SPBP is also shown to repress the transcriptional activity of estrogenreceptor α (ERα). Over-expression of SPBP inhibited the proliferation ofan ERα-dependent breast cancer cell line (Gburcik et al., 2005). In thecell nucleus, SPBP displays relatively low mobility and is enriched inchromatin dense regions, clearly indicating that it is a chromatinbinding protein (Darvekar et al., 2012). TCF20 is important for enhancedinduction of proteins involved in the cellular defensive program againstoxidative stress (Darvekar et al., 2014).

C3 is a prominent element of the inflammatory tumor microenvironment(Rutkowski et al., 2010) and activation can give a tumor growthadvantage (Markiewski et al., 2008). Enzymatic cleavage of C3 leads tothe production of the anaphylatoxin C3a, an inflammatory mediator andchemoattractant, and C3b (Sahu et al., 1998).

CLN3 is an anti-apoptotic gene in NT2 neuronal precursor cells and a fewtypes of cancers (Zhu et al., 2014b). It is involved in intracellulartrafficking and regulation in neuronal and non-neuronal cells (Rakhejaet al., 2008; Getty and Pearce, 2011) and it is implicated in severalimportant signaling pathways (Persaud-Sawin et al., 2002). CLN3 mRNA andprotein are over-expressed in a number of cancer cell lines includingbreast, colon, malignant melanoma, prostate, ovarian, neuroblastoma, andglioblastoma multiforme, but not lung or pancreatic cancer cell lines(Rylova et al., 2002).

SLC13A5 is one of 7 CIMP-marker genes. CIMP (CpG island methylatorphenotype) of clear cell renal cell carcinomas (ccRCCs) is characterizedby accumulation of DNA methylation at CpG islands and poorer patientoutcome (Tian et al., 2014; Arai et al., 2012).

SLC35B2 is involved in coordinated transcriptional regulation duringinduction of sialyl sulfo-Lex glycan biosynthesis during acuteinflammation (Huopaniemi et al., 2004) and in the sulfation of the6-sulfolactosamine epitope in a human colorectal carcinoma cell line(Kamiyama et al., 2006). Colorectal carcinoma cell lines as well ashuman colorectal tissues express SLC35B2 (Kamiyama et al., 2011).

PLOD1 expression is associated with human breast cancer progression(Gilkes et al., 2013).

PRDX5 is up-regulated in many malignant tumors (Urig and Becker, 2006)and inhibition of PRDX5 could prevent the tumor initiation andprogression, suggesting PRDX5 to be a promising target for cancertherapy. Its highly nucleophilic and accessible selenocysteine activesite might be the prime target for drug design (Liu et al., 2012).

Increased expression of PSMD8 in the peripheral lung may be potentiallyinformative as to what critical cell populations are involved in thedevelopment of invasive cancers (Zhou et al., 1996).

SNRPD1 is a core spliceosomal protein, which is up-regulated inmalignant tumors.

Reduced expression of the SPTBN1 is associated with worsened prognosisin pancreatic cancer (Jiang et al., 2010).

SQSTM1 functions as a signaling hub for various signal transductionpathways, such as NF-κB signaling, apoptosis, and Nrf2 activation, whosedysregulation is associated with Paget disease of bone and tumorigenesis(Komatsu et al., 2012).

PCNA expression predicts survival in anorectal malignant melanoma(Ben-Izhak et al., 2002). A cancer-associated isoform of PCNA (caPCNA)was identified that contained an unusual pattern of methyl ester groupson numerous glutamic and aspartic acid residues within PCNA (Hoelz etal., 2006).

Depleting SRP54 in several tumor cell lines did not produce overtcellular phenotypes, such as growth arrest or death, even in cellsselected for stable reduction of SRP components (Ren et al., 2004).

At the molecular level, STAT1 inhibits the proliferation of both mouseand human tumor cells treated with IFN-γ via its ability to increase theexpression of cyclin-dependent kinase inhibitor p21Cip1, or to decreasec-myc expression (Ramana et al., 2000). The anti-tumor activity of STAT1is further supported by its ability to inhibit angiogenesis and tumormetastasis in mouse models (Huang et al., 2002). Increased STAT1 mRNAlevels were shown to be part of a molecular signature associated withbetter prediction of the metastatic outcome for patients with hormonereceptor negative and triple-negative breast cancers (Yau et al., 2010).

Fine-needle aspirate samples from follicular neoplasms demonstrated thatmalignant nodules over-express STT3A as compared with benign disease(Patel et al., 2011).

A meta-analysis showed that the STXBP4/COX11 rs6504950 polymorphism issignificantly correlated with breast cancer risk (Tang et al., 2012).

A peptide consisting or consisting essentially of the amino acidsequence as indicated herein can have one or two non-anchor amino acids(see below regarding the anchor motif) exchanged without that theability to bind to a molecule of the human major histocompatibilitycomplex (MHC) class-I or -II is substantially changed or is negativelyaffected, when compared to the non-modified peptide. In anotherembodiment, in a peptide consisting essentially of the amino acidsequence as indicated herein, one or two amino acids can be exchangedwith their conservative exchange partners (see herein below) withoutthat the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II is substantiallychanged, or is negatively affected, when compared to the non-modifiedpeptide.

The present invention further relates to a peptide according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds as described herein below.

The present invention further relates to a peptide according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells, i.e. binds to dendritic cells.

The present invention further relates to a nucleic acid, encoding for apeptide according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing, expressing, and/or presenting a nucleic acid according tothe present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine.

The present invention further relates to antibodies as described furtherbelow, and methods of making them. Preferred are antibodies that arespecific for the peptides of the present invention, and/or for thepeptides of the present invention when bound to their MHC. Preferredantibodies can be monoclonal.

The present invention further relates to T-cell receptors (TCR), inparticular soluble TCR (sTCRs) targeting the peptides according to theinvention and/or the peptide-MHC complexes thereof, and methods ofmaking them.

The present invention further relates to antibodies or other bindingmolecules targeting the peptides according to the invention and/or thepeptide-MHC complexes thereof, and methods of making them.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell. The present invention further relates to the host cell accordingto the present invention, wherein the antigen presenting cell is adendritic cell.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature cited therein) are shortsingle-stranded nucleic acid or peptide molecules, which can fold intodefined three-dimensional structures and recognize specific targetstructures. They have appeared to be suitable alternatives fordeveloping targeted therapies. Aptamers have been shown to selectivelybind to a variety of complex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumour cell lineshave been identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumours, while non-tumourgenic and primary healthy cells are notrecognized. If the identified aptamers recognise not only a specifictumour sub-type but rather interact with a series of tumours, thisrenders the aptamers applicable as so-called broad-spectrum diagnosticsand therapeutics.

Further, investigation of cell-binding behaviour with flow cytometryshowed that aptamers revealed very good apparent affinities in thenanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumour cellsand thus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumour cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 300,according to the present invention with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (eg antigenic) determinant. In one embodiment,a scaffold is able to direct the entity to which it is attached (e.g. a(second) antigen binding moiety) to a target site, for example to aspecific type of tumor cell or tumor stroma bearing the antigenicdeterminant (e.g. the complex of a peptide according to the applicationat hand). In another embodiment a scaffold is able to activate signalingthrough its target antigen, for example a T cell receptor complexantigen. Scaffolds include but are not limited to antibodies andfragments thereof, antigen binding domains of an antibody, comprising anantibody heavy chain variable region and an antibody light chainvariable region, binding proteins comprising at least one ankyrin repeatmotif and Single domain antigen binding (SDAB) molecules, apatmers,(soluble) TCRs and (modified) cells such as allogenic or autologous Tcells.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualisation of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, anti-CD28. Polypeptide scaffolds aredescribed, for example, in the background section of WO 2014/071978A1,and the references as cited therein.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom the host cell and/or its culture medium.

The present invention further relates to an in vitro method forproducing activated T-cells, the method comprising contacting in vitro Tcells with antigen loaded human class I or II MHC molecules expressed onthe surface of a suitable antigen-presenting cell for a period of timesufficient to activate said T cells in an antigen specific manner,wherein said antigen is at least one peptide according to the presentinvention. The present invention further relates to a method, whereinthe antigen is loaded onto class I or II MHC molecules expressed on thesurface of a suitable antigen-presenting cell by contacting a sufficientamount of the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing said peptide containing SEQ IDNO: 1 to SEQ ID NO: 300, or a variant amino acid sequence thereof.

The present invention further relates to activated T cells, produced bythe method according to the present invention, which selectivelyrecognize a cell which aberrantly expresses a polypeptide comprising anamino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated T-cell according to thepresent invention as a medicament or in the manufacture of a medicament.

The present invention further relates to a use according to the presentinvention, wherein said medicament is a vaccine, a cell, a cellpopulation, such as, for example, a cell line, sTCRs and monoclonalantibodies.

The present invention further relates to a use according to the presentinvention, wherein the medicament is active against cancer.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are cells of HCC.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention thatcan be used in the diagnosis and/or prognosis of HCC.

Furthermore, the present invention relates to the use of these noveltargets for cancer treatment.

Further, the present invention relates to a method for producing apersonalized anti-cancer vaccine for an individual patient using adatabase (herein designated also as “warehouse”) of pre-screened tumorassociated peptides.

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize Class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, or 13 and in case ofMHC class II peptides (elongated variants of the peptides of theinvention) they can be as long as 14, 15, 16, 17, 18, 19 or 20 aminoacids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “the peptides of the present invention” shall also include thepeptides consisting of or comprising a peptide as defined aboveaccording to SEQ ID NO: 1 to SEQ ID NO: 300.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 6 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori M, et al. HLA gene and haplotype frequencies in the North Americanpopulation: the National Marrow Donor Program Donor Registry.Transplantation. 1997 Oct. 15; 64(7): 1017-27) employing theHardy-Weinberg formula F = 1 − (1 − Gf)². Combinations of A*02 or A*24with certain HLA-DR alleles might be enriched or less frequent thanexpected from their single frequencies due to linkage disequilib- rium.For details refer to Chanock et al. (S. J. Chanock, et al (2004) HLA-A,-B, -Cw, -DQA1 and DRB1 in an African American popula- tion fromBethesda, USA Human Immunology, 65: 1223-1235). Calculated phenotypeAllele Population from allele frequency A*02 Caucasian (North America) 49.1% A*02 African American (North America)  34.1% A*02 Asian American(North America)  43.2% A*02 Latin American (North American)  48.3% DR1Caucasian (North America)  19.4% DR2 Caucasian (North America)  28.2%DR3 Caucasian (North America)  20.6% DR4 Caucasian (North America) 30.7% DR5 Caucasian (North America)  23.3% DR6 Caucasian (NorthAmerica)  26.7% DR7 Caucasian (North America)  24.8% DR8 Caucasian(North America)  5.7% DR9 Caucasian (North America)  2.1% DR1 African(North) American 13.20% DR2 African (North) American 29.80% DR3 African(North) American 24.80% DR4 African (North) American 11.10% DR5 African(North) American 31.10% DR6 African (North) American 33.70% DR7 African(North) American 19.20% DR8 African (North) American 12.10% DR9 African(North) American  5.80% DR1 Asian (North) American  6.80% DR2 Asian(North) American 33.80% DR3 Asian (North) American  9.20% DR4 Asian(North) American 28.60% DR5 Asian (North) American 30.00% DR6 Asian(North) American 25.10% DR7 Asian (North) American 13.40% DR8 Asian(North) American 12.70% DR9 Asian (North) American 18.60% DR1 Latin(North) American 15.30% DR2 Latin (North) American 21.20% DR3 Latin(North) American 15.20% DR4 Latin (North) American 36.80% DR5 Latin(North) American 20.00% DR6 Latin (North) American 31.10% DR7 Latin(North) American 20.20% DR8 Latin (North) American 18.60% DR9 Latin(North) American  2.10% A*24 Philippines   65% A*24 Russia Nenets   61%A*24:02 Japan   59% A*24 Malaysia   58% A*24:02 Philippines   54% A*24India   47% A*24 South Korea   40% A*24 Sri Lanka   37% A*24 China   32%A*24:02 India   29% A*24 Australia West   22% A*24 USA   22% A*24 RussiaSamara   20% A*24 South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02 or A*24. A vaccine mayalso include pan-binding MHC class II peptides. Therefore, the vaccineof the invention can be used to treat cancer in patients that are eitherA*02 positive, A*24 positive or positive for A*02 and A*24, whereas noselection for MHC class II allotypes is necessary due to the pan-bindingnature of these peptides.

Combining for example A*02 and A*24 peptides in one vaccine has theadvantage that a higher percentage of any patient population can betreated compared with addressing either MHC class I allele alone. Whilein most populations less than 50% of patients could be addressed byeither allele alone, the vaccine of the invention can treat at least 60%of patients in any relevant population. Specifically, the followingpercentages of patients will be positive for at least one of thesealleles in various regions: USA 61%, Western Europe 62%, China 75%,South Korea 77%, Japan 86% (calculated from www.allelefrequencies.net).

As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence. The term “codingregion” refers to that portion of a gene which either naturally ornormally codes for the expression product of that gene in its naturalgenomic environment, i.e., the region coding in vivo for the nativeexpression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding (or encoding) for apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly contemplated.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform.

The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment,” when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent homology”,“percent identity” or “percent identical”, when referring to a sequence,means that a sequence is compared to a claimed or described sequenceafter alignment of the sequence to be compared (the “Compared Sequence”)with the described or claimed sequence (the “Reference Sequence”). ThePercent Identity is then determined according to the following formula:

Percent Identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences; and R is the number of bases or amino acids in the ReferenceSequence over the length of the alignment with the Compared Sequencewith any gap created in the Reference Sequence also being counted as abase or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the Percent Identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum Percent Identity to theReference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified PercentIdentity.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,amino acids possessing non-standard R groups (i.e., R groups other thanthose found in the common 20 amino acids of natural proteins) may alsobe used for substitution purposes to produce immunogens and immunogenicpolypeptides according to the present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would simultaneously be substituted.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4.

Combinations of the elongations according to the invention can bedepicted from Table 7:

C-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 C-terminusN-terminus 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted CTLs, effector functions may be lysisof peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 μM, andmost preferably no more than about 10 μM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

MHC class I molecules can be found on most cells having a nucleus whichpresent peptides that result from proteolytic cleavage of mainlyendogenous, cytosolic or nuclear proteins, DRIPS, and larger peptides.However, peptides derived from endosomal compartments or exogenoussources are also frequently found on MHC class I molecules. Thisnon-classical way of class I presentation is referred to ascross-presentation in literature.

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by eitherCD8-positive T cells (MHC class I molecule) or by CD4-positive T cells(MHC class II molecule) is important in the development of tumorvaccines. It is therefore an object of the present invention, to providecompositions of peptides that contain peptides binding to MHC complexesof either class.

Considering the severe side-effects and expense associated with treatingcancer better prognosis and diagnostic methods are desperately needed.Therefore, there is a need to identify other factors representingbiomarkers for cancer in general and HCC in particular. Furthermore,there is a need to identify factors that can be used in the treatment ofcancer in general and HCC in particular.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably HCC that over- or exclusively present thepeptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanHCC samples.

The source gene/protein (also designated “full-length protein” or“underlying protein”) from which the peptides are derived were shown tobe highly overexpressed in cancer compared with normal tissues—“normaltissues” in relation to this invention shall mean either healthy livercells or other normal tissue cells, demonstrating a high degree of tumorassociation of the source genes (see example 2). Moreover, the peptidesthemselves are strongly over-presented on tumor tissue—“tumor tissue” inrelation to this invention shall mean a sample from a patient sufferingfrom HCC, but not on normal tissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. HCC cells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention (see Example 3). Furthermore, thepeptides when complexed with the respective MHC can be used for theproduction of antibodies and/or TCRs, in particular sTCRs, according tothe present invention, as well. Respective methods are well known to theperson of skill, and can be found in the respective literature as well.Thus, the peptides of the present invention are useful for generating animmune response in a patient by which tumor cells can be destroyed. Animmune response in a patient can be induced by direct administration ofthe described peptides or suitable precursor substances (e.g. elongatedpeptides, proteins, or nucleic acids encoding these peptides) to thepatient, ideally in combination with an agent enhancing theimmunogenicity (i.e. an adjuvant). The immune response originating fromsuch a therapeutic vaccination can be expected to be highly specificagainst tumor cells because the target peptides of the present inventionare not presented on normal tissues in comparable copy numbers,preventing the risk of undesired autoimmune reactions against normalcells in the patient.

A “pharmaceutical composition” preferably is preferably a compositionsuitable for administration to a human being in a medical setting.Preferably, a pharmaceutical composition is sterile and producedaccording to GMP guidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Especially preferred is a composition and/or the use of saidcomposition, e.g. in the form of a vaccine, comprising the peptideshaving a sequence according to the SEQ ID NOs 1, 2, 7, 225, 228, 301,303, and 312 or a scaffold reactive against the peptides having asequence according to the SEQ ID NOs 1, 2, 7, 225, 228, 301, 303, and312 and their complexes to MHC molecules.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from HCC cells and since it was determined that thesepeptides are not or at lower levels present in normal tissues, thesepeptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for HCC. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate markers in immunotherapy approaches aiming to inducelymphocyte responses by different means, e.g. vaccination of protein,nucleic acids, autologous materials, adoptive transfer of lymphocytes.In gene therapy settings, lymphocyte responses against peptides can beconsidered in the assessment of side effects. Monitoring of lymphocyteresponses might also be a valuable tool for follow-up examinations oftransplantation therapies, e.g. for the detection of graft versus hostand host versus graft diseases.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Yet another aspect of the present invention then relates to a method ofproducing said antibody specifically binding to a human majorhistocompatibility complex (MHC) class I or II being complexed with aHLA-restricted antigen, the method comprising: immunizing a geneticallyengineered non-human mammal comprising cells expressing said human majorhistocompatibility complex (MHC) class I or II with a soluble form of aMHC class I or II molecule being complexed with said HLA-restrictedantigen; isolating mRNA molecules from antibody producing cells of saidnon-human mammal; producing a phage display library displaying proteinmolecules encoded by said mRNA molecules; and isolating at least onephage from said phage display library, said at least one phagedisplaying said antibody specifically bindable to said human majorhistocompatibility complex (MHC) class I or II being complexed with saidHLA-restricted antigen. Respective methods for producing such antibodiesand single chain class I major histocompatibility complexes, as well asother tools for the production of these antibodies are disclosed in WO03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, and Cohen C J, etal. Recombinant antibodies with MHC-restricted, peptide-specific, T-cellreceptor-like specificity: new tools to study antigen presentation andTCR-peptide-MHC interactions. J Mol Recognit. 2003 September-October;16(5):324-32; Denkberg G, et al. Selective targeting of melanoma andAPCs using a recombinant antibody with TCR-like specificity directedtoward a melanoma differentiation antigen. J Immunol. 2003 Sep. 1;171(5):2197-207; and Cohen C J, et al. Direct phenotypic analysis ofhuman MHC class I antigen presentation: visualization, quantitation, andin situ detection of human viral epitopes using peptide-specific,MHC-restricted human recombinant antibodies. J Immunol. 2003 Apr. 15;170(8):4349-61, which for the purposes of the present invention are allexplicitly incorporated by reference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isregarded as “specific” in the context of the present invention.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, Liddy N, et al. Monoclonal TCR-redirected tumor cellkilling. Nat Med 2012 June; 18(6):980-987). For the purpose ofstabilization of T-cell receptors during phage display and in case ofpractical use as drug, alpha and beta chain can be linked e.g. bynon-native disulfide bonds, other covalent bonds (single-chain T-cellreceptor), or by dimerization domains (see Boulter J M, et al. Stable,soluble T-cell receptor molecules for crystallization and therapeutics.Protein Eng 2003 September; 16(9):707-711; Card K F, et al. A solublesingle-chain T-cell receptor IL-2 fusion protein retains MHC-restrictedpeptide specificity and IL-2 bioactivity. Cancer Immunol Immunother 2004April; 53(4):345-357; and Willcox B E, et al. Production of solublealphabeta T-cell receptor heterodimers suitable for biophysical analysisof ligand binding. Protein Sci 1999 November; 8 (11):2418-2423). TheT-cell receptor can be linked to toxins, drugs, cytokines (see, forexample, US 2013/0115191), domains recruiting effector cells such as ananti-CD3 domain, etc., in order to execute particular functions ontarget cells. Moreover, it could be expressed in T cells used foradoptive transfer. Further information can be found in WO 2004/033685A1and WO 2004/074322A1. A combination of sTCRs is described in WO2012/056407A1. Further methods for the production are disclosed in WO2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (J. Pinheiro, et al. Thenlme Package: Linear and Nonlinear Mixed Effects Models. 2007) adjustingfor multiple testing by False Discovery Rate (Y. Benjamini and Y.Hochberg. Controlling the False Discovery Rate: A Practical and PowerfulApproach to Multiple Testing. Journal of the Royal Statistical Society.Series B (Methodological), Vol. 57 (No. 1):289-300, 1995).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of natural TUMAPsrecorded from HCC samples (N=16 A*02-positive samples including thirteenA*02:01-positive samples, N=15 A*24-positive samples) with thefragmentation patterns of corresponding synthetic reference peptides ofidentical sequences. Since the peptides were directly identified asligands of HLA molecules of primary tumors, these results provide directevidence for the natural processing and presentation of the identifiedpeptides on primary cancer tissue obtained from 31 HCC patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from HCC tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on primary HCC samples confirming theirpresentation on primary HCC.

TUMAPs identified on multiple HCC tumor and normal tissues werequantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 300, ora variant thereof which is at least 90% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 300 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:300 or a variant thereof which is at least 90% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 300, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 300.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of HCC.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to the method according to thepresent invention, where—in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 300 or said variant amino acid sequence.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are HCC cells or other solid orhaematological tumor cells such as pancreatic cancer, brain cancer,kidney cancer, colon or rectal cancer, or leukemia.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of HCC. The present invention also relates to the use of thesenovel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a HCC marker polypeptide, deliveryof a toxin to a HCC cell expressing a cancer marker gene at an increasedlevel, and/or inhibiting the activity of a HCC marker polypeptide)according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length HCC marker polypeptides or fragments thereof maybe used to generate the antibodies of the invention. A polypeptide to beused for generating an antibody of the invention may be partially orfully purified from a natural source, or may be produced usingrecombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 300polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the HCC marker polypeptide used togenerate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1988, new 2nd edition 2013). For example, the antibodies may be testedin ELISA assays or, Western blots, immunohistochemical staining offormalin-fixed cancers or frozen tissue sections. After their initial invitro characterization, antibodies intended for therapeutic or in vivodiagnostic use are tested according to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating HCC, the efficacyof the therapeutic antibody can be assessed in various ways well knownto the skilled practitioner. For instance, the size, number, and/ordistribution of cancer in a subject receiving treatment may be monitoredusing standard tumor imaging techniques. A therapeutically-administeredantibody that arrests tumor growth, results in tumor shrinkage, and/orprevents the development of new tumors, compared to the disease coursethat would occurs in the absence of antibody administration, is anefficacious antibody for treatment of cancer.

Because the peptides as mentioned in the Tables above of the inventionand thus their underlying polypeptides are highly expressed in HCC, andare expressed at rather to extremely low levels in normal cells, theinhibition of a protein selected from the group consisting of proteinproducts of the following genes: Preferred for the inhibition and forantibodies and/or TCRs against are GLUL, GPAM, PLIN2, SLC16A1, SLC9A3R1,PCBD1, SEC16A, AKR1C4, ABCB11, HAL, CYP2E1, C4A, C4B, ALDH1L1, CRP,ACSL4, EEF2, HLTF, FBXO22, GALK1, TMCO1, TMEM33, ZNF318, IPO9, AMACR,C1QTNF3, CYP4F8, CYP4F3, CYP4F11, CYP4F12, CYP4F2, MOCOS, A1CF, COL18A1,HPR, LBP, C19orf80, CFHR5, ITIH4, TMEM110, LARP4, LMF2, SLC10A5, andSLC16A11; still preferred for the inhibition and for antibodies and/orTCRs against are ANKFY1, C12orf44, C16orf58, CPSF1, DCAF8, PEX19, DDX11,DDX12P, DECR2, NME4, DENND5B, DYM, EDC4, ERI3, FAM20A, FNDC3A, GPR107,GYG2, HEATR2, IFT81, KCTD3, SHKBP1, KIAA1324L, KLHL24, MARCH6, MBTPS2,MIR1279, CPSF6, NOC4L, NXF1, PANK2, PCNXL3, PIPSL, PSMD4, PSMD14,SLC35B1, TCP11L2, THNSL2, THOC2, TOMM5, TRAPPC6B, TRIM54, TRIM55,TRIM63, UGGT2, URB1, VPS54, WIZ, ZNF451, RFTN2, SCFD1, SERINC5, CCT7P2,CMAS, ANKS1A, C17orf70, CCT7, CDK5RAP2, CLPTM1, and most preferred forthe inhibition and for antibodies and/or TCRs against are APOB, FASN,and/or COPA; and expression or of the activity of these markers may bepreferably integrated into a therapeutic strategy, e.g. for treating orpreventing HCC.

The principle of antisense therapy is based on the hypothesis thatsequence-specific suppression of gene expression (via transcription ortranslation) may be achieved by intra-cellular hybridization betweengenomic DNA or mRNA and a complementary antisense species. The formationof such a hybrid nucleic acid duplex interferes with transcription ofthe target tumor antigen-encoding genomic DNA, orprocessing/transport/translation and/or stability of the target tumorantigen mRNA.

Antisense nucleic acids can be delivered by a variety of approaches. Forexample, antisense oligonucleotides or anti-sense RNA can be directlyadministered (e.g., by intravenous injection) to a subject in a formthat allows uptake into tumor cells. Alternatively, viral or plasmidvectors that encode antisense RNA (or RNA fragments) can be introducedinto cells in vivo. Antisense effects can also be induced by sensesequences; however, the extent of phenotypic changes is highly variable.Phenotypic changes induced by effective antisense therapy are assessedaccording to changes in, e.g., target mRNA levels, target proteinlevels, and/or target protein activity levels.

In a specific example, inhibition of HCC target/marker function byantisense gene therapy may be accomplished by direct administration ofantisense tumor marker RNA to a subject. The antisense tumor marker RNAmay be produced and isolated by any standard technique, but is mostreadily produced by in vitro transcription using an antisense tumormarker cDNA under the control of a high efficiency promoter (e.g., theT7 promoter). Administration of anti-sense tumor marker RNA to cells canbe carried out by any of the methods for direct nucleic acidadministration described below.

An alternative strategy for inhibiting the function of a proteinselected from the group consisting of the above-mentioned proteins, andmost preferred of APOB, FASN, and/or COPA, involves use of a nucleicacid (e.g. siRNA, or a nucleic acid coding for an anti-protein antibodyor a portion thereof, which can be transferred into cancer cells orother cells, leading to intracellular antibody expression andsecretion), a protein or small molecule, or any other compound targetingthe expression, translation, and/or biological function of this protein.

In the methods described above, which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of naked DNA or the nucleic acids can be ina vector for delivering the nucleic acids to the cells for inhibition ofHCC marker protein expression. The vector can be a commerciallyavailable preparation, such as an adenovirus vector (QuantumBiotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleicacid or vector to cells can be via a variety of mechanisms. As oneexample, delivery can be via a liposome, using commercially availableliposome preparations such as Lipofectin, Lipofectamine (GIBCO-25 BRL,Inc., Gaithersburg, Md.), Superfect (Qiagen, Inc. Hilden, Germany) andTransfectam (Promega Biotec, Inc., Madison, Wis., US), as well as otherliposomes developed according to procedures standard in the art. Inaddition, the nucleic acid or vector of this invention can be deliveredin vivo by electroporation, the technology for which is available fromGenetronics, Inc. (San Diego, US) as well as by means of a Sonoporationmachine (ImaRx Pharmaceutical Corp., Tucson, Ariz., US).

As one example, vector delivery can be via a viral system, such as aretroviral vector system that can package a recombinant retroviralgenome. The recombinant retrovirus can then be used to infect andthereby deliver to the infected cells antisense nucleic acid thatinhibits expression of a protein selected from the group consisting ofthe above-mentioned proteins. The exact method of introducing thealtered nucleic acid into mammalian cells is, of course, not limited tothe use of retroviral vectors. Other techniques are widely available forthis procedure including the use of adenoviral vectors, adeno-associatedviral (AAV) vectors, lentiviral vectors, pseudotyped retroviral vectors.Physical transduction techniques can also be used, such as liposomedelivery and receptor-mediated and other endocytosis mechanisms. Thisinvention can be used in conjunction with any of these or other commonlyused gene transfer methods.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹TC, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S)) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 300 or a variant thereof which is 90%homologous to SEQ ID NO: 1 to SEQ ID NO: 300, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see Percent Identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or otheranalysis tools are provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Fong L, et al. Altered peptide ligandvaccination with Flt3 ligand expanded dendritic cells for tumorimmunotherapy. Proc Natl Acad Sci USA. 2001 Jul. 17; 98(15):8809-14;Zaremba S, et al. Identification of an enhancer agonist cytotoxic Tlymphocyte peptide from human carcinoembryonic antigen. Cancer Res. 1997Oct. 15; 57(20):4570-7; Colombetti S, et al. Impact of orthologousmelan-A peptide immunizations on the anti-self melan-A/HLA-A2 T cellcross-reactivity. J Immunol. 2006 Jun. 1; 176(11):6560-7; Appay V, etal. Decreased specific CD8+ T cell cross-reactivity of antigenrecognition following vaccination with Melan-A peptide. Eur J Immunol.2006 July; 36(7):1805-14).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 300. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature (Godkin A, et al. Use ofeluted peptide sequence data to identify the binding characteristics ofpeptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8(DQ 3.2). Int Immunol. 1997 June; 9(6):905-11) and databases (RammenseeH. et al. SYFPEITHI: database for MHC ligands and peptide motifs.Immunogenetics. 1999 November; 50(3-4):213-9), certain positions of HLAbinding peptides are typically anchor residues forming a core sequencefitting to the binding motif of the HLA receptor, which is defined bypolar, electrophysical, hydrophobic and spatial properties of thepolypeptide chains constituting the binding groove. Thus, one skilled inthe art would be able to modify the amino acid sequences set forth inSEQ ID NO: 1 to SEQ ID NO 300, by maintaining the known anchor residues,and would be able to determine whether such variants maintain theability to bind MHC class I or II molecules. The variants of the presentinvention retain the ability to bind to the TCR of activated T cells,which can subsequently cross-react with and kill cells that express apolypeptide containing the natural amino acid sequence of the cognatepeptide as defined in the aspects of the invention.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith another amino acid whose incorporation does not substantiallyaffect T-cell reactivity and does not eliminate binding to the relevantMHC. Thus, apart from the proviso given, the peptide of the inventionmay be any peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

Those amino acid residues that do not substantially contribute tointeractions with the TCR can be modified by replacement with anotheramino acid whose incorporation does not substantially affect T-cellreactivity and does not eliminate binding to the relevant MHC. Thus,apart from the proviso given, the peptide of the invention may be anypeptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 8  Variants and motif of the peptidesaccording to SEQ ID NO: 1, 117, and 246 Position 1 2 3 4 5 6 7 8 9SEQ ID NO. 1 V M A P F T M T I Variants V L A L V L L L L A A V A L A AA V V V L V V A T V T L T T A Q V Q L Q Q A Position 1 2 3 4 5 6 7 8 9SEQ ID NO 246 K L I S S Y Y N V Variants I L I I I I A M L M I M M A A LA I A A A V L V I V V A T L T I T T A Q L Q I Q Q A Position 1 2 3 4 5 67 8 9 SEQ ID NO. 117 Y A F P K S I T V Variants L I A M L M I M M A L LL I L L A V L V I V V A T L T I T T A Q L Q I Q Q A

Longer peptides may also be suitable. It is also possible that MHC classI epitopes, although usually between 8 and 11 amino acids long, aregenerated by peptide processing from longer peptides or proteins thatinclude the actual epitope. It is preferred that the residues that flankthe actual epitope are residues that do not substantially affectproteolytic cleavage necessary to expose the actual epitope duringprocessing.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 300.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 300 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) J. Immunol. 159,3230-3237, incorporated herein by reference. This approach involvesmaking pseudopeptides containing changes involving the backbone, and notthe orientation of side chains. Meziere et al (1997) show that for MHCbinding and T helper cell responses, these pseudopeptides are useful.Retro-inverse peptides, which contain NH—CO bonds instead of CO—NHpeptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2005, which is incorporatedherein by reference. Chemical modification of amino acids includes butis not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) for more extensivemethodology relating to chemical modification of proteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich(http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Solid-phase peptide synthesis under continuous-flow conditions. ProcNatl Acad Sci USA. May 1981; 78(5): 2791-2795), and references as citedtherein. Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group.

Repetitive cleavage of this highly base-labile protecting group is doneusing 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethandithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, Bruckdorfer et al., 2004, and the references as citedtherein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitril/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Diagnosis of sickle cell anemia and beta-thalassemia withenzymatically amplified DNA and nonradioactive allele-specificoligonucleotide probes. N Engl J Med. 1988 Sep. 1; 319(9):537-41). Thismethod may be used for introducing the DNA into a suitable vector, forexample by engineering in suitable restriction sites, or it may be usedto modify the DNA in other useful ways as is known in the art. If viralvectors are used, pox- or adenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasm ids pRS413-416 are Yeast Centromere plasmids(Ycps). CMV promoter-based vectors (for example from Sigma-Aldrich)provide transient or stable expression, cytoplasmic expression orsecretion, and N-terminal or C-terminal tagging in various combinationsof FLAG, 3×FLAG, c-myc or MAT. These fusion proteins allow fordetection, purification and analysis of recombinant protein. Dual-taggedfusions provide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl.Acad. Sci. USA 69, 2110, and Sambrook et al (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Transformation of yeast cells is described in Sherman et al (1986)Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y.The method of Beggs (1978) Nature 275, 104-109 is also useful. Withregard to vertebrate cells, reagents useful in transfecting such cells,for example calcium phosphate and DEAE-dextran or liposome formulations,are available from Stratagene Cloning Systems, or Life TechnologiesInc., Gaithersburg, Md. 20877, USA. Electroporation is also useful fortransforming and/or transfecting cells and is well known in the art fortransforming yeast cell, bacterial cells, insect cells and vertebratecells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Small E J, et al. Placebo-controlled phase IIItrial of immunologic therapy with sipuleucel-T (APC8015) in patientswith metastatic, asymptomatic hormone refractory prostate cancer. J ClinOncol. 2006 Jul. 1; 24(19):3089-94. Rini et al. Combinationimmunotherapy with prostatic acid phosphatase pulsed antigen-presentingcells (provenge) plus bevacizumab in patients with serologic progressionof prostate cancer after definitive local therapy. Cancer. 2006 Jul. 1;107(1):67-74).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al Nature Medicine 18, 1254-1261 (2012)).

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Karre et al.(Ljunggren, H.-G., and K. Karre. 1985. J. Exp. Med. 162:1745).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 300, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Induction of peptide-specificprimary cytotoxic T lymphocyte responses from human peripheral blood.Eur J Immunol. 1995 June; 25(6):1783-7) make use of autologousperipheral blood lymphocytes (PLBs) in the preparation of T cells.Furthermore, the production of autologous T cells by pulsing dendriticcells with peptide or polypeptide, or via infection with recombinantvirus is possible. Also, B cells can be used in the production ofautologous T cells. In addition, macrophages pulsed with peptide orpolypeptide, or infected with recombinant virus, may be used in thepreparation of autologous T cells. S. Walter et al. 2003 (Cutting edge:predetermined avidity of human CD8 T cells expanded on calibratedMHC/anti-CD28-coated microspheres. J Immunol. 2003 Nov. 15;171(10):4974-8) describe the in vitro priming of T cells by usingartificial antigen presenting cells (aAPCs), which is also a suitableway for generating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e.g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition plant viruses may be used (see, for example,Porta et al. (1994) Development of cowpea mosaic virus as ahigh-yielding system for the presentation of foreign peptides. Virology.1994 Aug. 1; 202(2):949-55) which describes the development of cowpeamosaic virus as a high-yielding system for the presentation of foreignpeptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 300.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattinoni L, et al. Adoptiveimmunotherapy for cancer: building on success. Nat Rev Immunol. 2006May; 6(5):383-93. Review. and Morgan R A, et al. Cancer regression inpatients after transfer of genetically engineered lymphocytes. Science.2006 Oct. 6; 314(5796):126-9).

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

Preferably, the medicament of the present invention is a vaccine. It maybe administered directly into the patient, into the affected organ orsystemically i.d., i.m., s.c., i.p. and i.v., or applied ex vivo tocells derived from the patient or a human cell line which aresubsequently administered to the patient, or used in vitro to select asubpopulation of immune cells derived from the patient, which are thenre-administered to the patient. If the nucleic acid is administered tocells in vitro, it may be useful for the cells to be transfected so asto co-express immune-stimulating cytokines, such as interleukin-2. Thepeptide may be substantially pure, or combined with animmune-stimulating adjuvant (see below) or used in combination withimmune-stimulatory cytokines, or be administered with a suitabledelivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see for example WO 95/18145). The peptide may also betagged, may be a fusion protein, or may be a hybrid molecule. Thepeptides whose sequence is given in the present invention are expectedto stimulate CD4 or CD8 T cells. However, stimulation of CD8 T cells ismore efficient in the presence of help provided by CD4 T-helper cells.Thus, for MHC Class I epitopes that stimulate CD8 T cells the fusionpartner or sections of a hybrid molecule suitably provide epitopes whichstimulate CD4-positive T cells. CD4- and CD8-stimulating epitopes arewell known in the art and include those identified in the presentinvention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 300, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

In another aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 300, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

The polynucleotide may be substantially pure, or contained in a suitablevector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNAor a combination thereof. Methods for designing and introducing such anucleic acid are well known in the art. An overview is provided by e.g.(Pascolo et al., Human peripheral blood mononuclear cells transfectedwith messenger RNA stimulate antigen-specific cytotoxic T-lymphocytes invitro. Cell Mol Life Sci. 2005 August; 62(15):1755-62). Polynucleotidevaccines are easy to prepare, but the mode of action of these vectors ininducing an immune response is not fully understood. Suitable vectorsand delivery systems include viral DNA and/or RNA, such as systems basedon adenovirus, vaccinia virus, retroviruses, herpes virus,adeno-associated virus or hybrids containing elements of more than onevirus. Non-viral delivery systems include cationic lipids and cationicpolymers and are well known in the art of DNA delivery. Physicaldelivery, such as via a “gene-gun” may also be used. The peptide orpeptides encoded by the nucleic acid may be a fusion protein, forexample with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995 The Yin and Yang of T cell costimulation. Science. 1995Nov. 10; 270(5238):932-3). Also cytokines may be used. Several cytokineshave been directly linked to influencing dendritic cell migration tolymphoid tissues (e.g., TNF-), accelerating the maturation of dendriticcells into efficient antigen-presenting cells for T-lymphocytes (e.g.,GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specificallyincorporated herein by reference in its entirety) and acting asimmunoadjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta)(Gabrilovich, 1996 Production of vascular endothelial growth factor byhuman tumors inhibits the functional maturation of dendritic cells NatMed. 1996 October; 2(10): 1096-103).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients, 3rd Ed., 2000, AmericanPharmaceutical Association and pharmaceutical press. The composition canbe used for a prevention, prophylaxis and/or therapy of adenomateous orcancerous diseases. Exemplary formulations can be found in, for example,EP2112253.

The present invention provides a medicament that useful in treatingcancer, in particular HCC and other malignancies.

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from HCC, themedicament of the invention is preferably used to treat HCC.

The present invention further includes a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group of peptidesthat have been pre-screened for immunogenicity and/or over-presentationin a particular tumor type. The term “warehouse” is not intended toimply that the particular peptides included in the vaccine have beenpre-manufactured and stored in a physical facility, although thatpossibility is contemplated. It is expressly contemplated that thepeptides may be manufactured de novo for each individualized vaccineproduced, or may be pre-manufactured and stored. The warehouse (e.g. inthe form of a database) is composed of tumor-associated peptides whichwere highly overexpressed in the tumor tissue of HCC patients withvarious HLA-A HLA-B and HLA-C alleles. It may contain MHC class I andMHC class II peptides or elongated MHC class I peptides. In addition tothe tumor associated peptides collected from several HCC tissues, thewarehouse may contain HLA-A*02 and HLA-A*24 marker peptides. Thesepeptides allow comparison of the magnitude of T-cell immunity induced byTUMAPS in a quantitative manner and hence allow important conclusion tobe drawn on the capacity of the vaccine to elicit anti-tumor responses.Secondly, they function as important positive control peptides derivedfrom a “non-self” antigen in the case that any vaccine-induced T-cellresponses to TUMAPs derived from “self” antigens in a patient are notobserved. And third, it may allow conclusions to be drawn, regarding thestatus of immunocompetence of the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®)). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, HCC samples from patients andblood from healthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (HCC)compared with a range of normal organs and tissues3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromHCC patients.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked.

This is an advantage over vaccines that address only one or few targets,which may cause the tumor to easily adapt to the attack (tumor escape).Furthermore, not all individual tumors express the same pattern ofantigens. Therefore, a combination of several tumor-associated peptidesensures that every single tumor bears at least some of the targets. Thecomposition was specifically designed in such a way that each HLA-A*02and/or HLA-A*24-positive tumor is expected to express several of theantigens and cover several independent pathways necessary for tumorgrowth and maintenance. For each of the peptide subsets specific for thetwo HLA class I alleles (A*02 and A*24) this is independently ensuredbased on the underlying experimental analyses. Thus, the vaccine caneasily be used “off-the-shelf” for a larger patient population. Thismeans that a pre-selection of patients to be treated with the vaccinecan be restricted to HLA typing, does not require any additionalbiomarker assessments for antigen expression, but it is still ensuredthat several targets are simultaneously attacked by the induced immuneresponse, which is important for efficacy (Banchereau et al., 2001;Walter et al., 2012).

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1)) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, nevertheless, withoutbeing limited thereto. For the purposes of the present invention, allreferences as cited herein are incorporated by reference in theirentireties.

In the Figures,

FIG. 1 shows the over-presentation of various peptides in normal tissues(dark gray) and HCC (light gray). FIG. 1A) APOB, Peptide: ALVDTLKFV(A*02) (SEQ ID NO: 7), tissues from left to right; 1 adipose tissues, 3adrenal glands, 2 arteries, 2 bone marrows, 7 brains, 3 breasts, 13colons, 4 esophagi, 2 gallbladders, 3 GI tracts, 3 hearts, 16 kidneys, 4leukocyte samples, 45 lungs, 1 lymph node, 1 ovary, 7 pancreas, 1peripheral nerve, 1 pituitary gland, 3 pleuras, 1 prostate, 6 recti, 3sceletal muscles, 1 serous membrane, 3 skins, 4 spleens, 7 stomachs, 1testis, 2 thymi, 3 thyroid glands, 2 uteri, 2 veins, and 20 livers; FIG.1B) ALDH1L1, Peptide: KLQAGTVFV (A*02) (SEQ ID NO: 2), tissues from leftto right: 1 adipose tissues, 3 adrenal glands, 2 arteries, 2 bonemarrows, 7 brains, 3 breasts, 13 colons, 4 esophagi, 2 gallbladders, 3GI tracts, 3 hearts, 16 kidneys, 4 leukocyte samples, 45 lungs, 1 lymphnode, 1 ovary, 7 pancreas, 1 peripheral nerve, 1 pituitary gland, 3pleuras, 1 prostate, 6 recti, 3 sceletal muscles, 1 serous membrane, 3skins, 4 spleens, 7 stomachs, 1 testis, 2 thymi, 3 thyroid glands, 2uteri, 2 veins, and 20 livers; FIG. 1C) C8B, Peptide: AYLLQPSQF (A*24)(SEQ ID NO: 200), tissues from left to right: including 2 adrenalglands, 1 artery, 4 brains, 1 breast, 5 colons, 1 hearts, 13 kidneys, 9lungs, 3 pancreas, 2 recti, 3 skins, 1 spleen, 12 stomachs, 1 thymus, 2uteri, and 9 livers; FIG. 1D) RAD23B Peptide: KIDEKNFVV (SEQ ID NO: 63)1 serous membrane, 1 adipose tissue, 3 adrenal glands, 2 arteries, 2bone marrows, 7 brains, 3 breasts, 13 colons, 2 gallbladders, 3 GItracts, 3 hearts, 12 kidneys, 4 leukocytes, 19 livers, 43 lungs, 1 lymphnode, 1 ovary, 6 pancreases, 1 peripheral nerve, 1 pituitary gland, 3pleuras, 1 prostate, 6 rectums, 3 skeletal muscles, 3 skins, 4 spleens,5 stomachs, 1 testis, 2 thymi, 3 thyroid glands, 2 uteri, 2 veins, 4esophagi; FIG. 1E) RAD23B Peptide: KIDEKNFVV (SEQ ID NO: 63), shown areonly samples on which the peptide was presented: 5 cell-lines, 1 normaltissue (1 adrenal gland), 16 cancer tissues (2 brain cancers, 4 livercancers, 5 lung cancers, 1 rectum cancer, 1 urinary bladder cancer, 3uterus cancers) (from left to right); FIG. 1F) RFNG RLPPDTLLQQV (SEQ IDNO: 92), shown are only samples on which the peptide was presented: 1serous membrane, 1 adipose tissue, 3 adrenal glands, 2 arteries, 2 bonemarrows, 7 brains, 3 breasts, 13 colons, 2 gallbladders, 3 GI tracts, 3hearts, 12 kidneys, 4 leukocytes, 19 livers, 43 lungs, 1 lymph node, 1ovary, 6 pancreases, 1 peripheral nerve, 1 pituitary gland, 3 pleuras, 1prostate, 6 rectums, 3 skeletal muscles, 3 skins, 4 spleens, 5 stomachs,1 testis, 2 thymi, 3 thyroid glands, 2 uteri, 2 veins, 4 esophagi; FIG.1G) RFNG Peptide: RLPPDTLLQQV (SEQ ID NO: 92), shown are only samples onwhich the peptide was presented: 2 cell-lines, 2 normal tissues (2adrenal glands), 17 cancer tissues (1 brain cancer, 1 breast cancer, 1esophageal cancer, 5 liver cancers, 4 lung cancers, 1 ovarian cancer, 1prostate cancer, 2 urinary bladder cancers, 1 uterus cancer) (from leftto right); FIG. 1H) FLVCR1 Peptide: SVWFGPKEV (SEQ ID NO: 104) 1 serousmembrane, 1 adipose tissue, 3 adrenal glands, 2 arteries, 2 bonemarrows, 7 brains, 3 breasts, 13 colons, 2 gallbladders, 3 GI tracts, 3hearts, 12 kidneys, 4 leukocytes, 19 livers, 43 lungs, 1 lymph node, 1ovary, 6 pancreases, 1 peripheral nerve, 1 pituitary gland, 3 pleuras, 1prostate, 6 rectums, 3 skeletal muscles, 3 skins, 4 spleens, 5 stomachs,1 testis, 2 thymi, 3 thyroid glands, 2 uteri, 2 veins, 4 esophagi; FIG.1I) FLVCR1 Peptide: SVWFGPKEV (SEQ ID NO: 104), shown are only sampleson which the peptide was presented: 9 cell lines, 1 normal tissue (1small intestine), 16 cancer tissues (1 brain cancer, 1 breast cancer, 5liver cancers, 5 lung cancers, 1 skin cancer, 1 stomach cancer, 1urinary bladder cancer, 1 uterus cancer) (from left to right); FIG. 1J)IKBKAP Peptide: LLFPHPVNQV (SEQ ID NO: 156) 1 serous membrane, 1 adiposetissue, 3 adrenal glands, 2 arteries, 2 bone marrows, 7 brains, 3breasts, 13 colons, 2 gallbladders, 3 GI tracts, 3 hearts, 12 kidneys, 4leukocytes, 19 livers, 43 lungs, 1 lymph node, 1 ovary, 6 pancreases, 1peripheral nerve, 1 pituitary gland, 3 pleuras, 1 prostate, 6 rectums, 3skeletal muscles, 3 skins, 4 spleens, 5 stomachs, 1 testis, 2 thymi, 3thyroid glands, 2 uteri, 2 veins, 4 esophagi; FIG. 1K) IKBKAP Peptide:LLFPHPVNQV (SEQ ID NO: 156), shown are only samples on which the peptidewas presented: 7 cell-lines, 2 primary cultures, 1 normal tissue (1colon), 34 cancer tissues (1 bone marrow cancer, 1 breast cancer, 1colon cancer, 2 esophageal cancers, 2 leukocytic leukemia cancers, 4liver cancers, 11 lung cancers, 3 lymph node cancers, 5 ovarian cancers,4 urinary bladder cancers) (from left to right); FIG. 1L) NKD1 Peptide:FLDTPIAKV (SEQ ID NO: 47), 1 serous membrane, 1 adipose tissue, 3adrenal glands, 2 arteries, 2 bone marrows, 7 brains, 3 breasts, 13colons, 2 gallbladders, 3 GI tracts, 3 hearts, 12 kidneys, 4 leukocytes,19 livers, 43 lungs, 1 lymph node, 1 ovary, 6 pancreases, 1 peripheralnerve, 1 pituitary gland, 3 pleuras, 1 prostate, 6 rectums, 3 skeletalmuscles, 3 skins, 4 spleens, 5 stomachs, 1 testis, 2 thymi, 3 thyroidglands, 2 uteri, 2 veins, 4 esophagi; FIG. 1M) NKD1 Peptide: FLDTPIAKV(SEQ ID NO: 47), shown are only samples on which the peptide waspresented: 1 other disease (encephalocele), 2 normal tissues (1 lung, 1spleen), 35 cancer tissues (5 brain cancers, 6 colon cancers, 1esophageal cancer, 6 liver cancers, 9 lung cancers, 1 ovarian cancer, 1prostate cancer, 4 rectum cancers, 2 stomach cancers) (from left toright).

FIG. 2 shows exemplary expression profiles (relative expression comparedto normal kidney) of source genes of the present invention that arehighly over-expressed or exclusively expressed in HCC in a panel ofnormal tissues (dark gray) and 12 HCC samples (gray). FIG. 2A) APOB,tissues from left to right: 1 adrenal gland, 1 artery, 1 bone marrow, 1brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 kidneys, 1leukocyte sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1 pancreas, 1placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1 skin, 1small intestine, 1 spleen, 1 stomach, 1 testis, 1 thymus, 1 thyroidgland, 1 urinary bladder, 1 uterine cervix, 1 uterus, 1 vein; FIG. 2B)AMACR, tissues from left to right: 1 adrenal gland, 1 artery, 1 bonemarrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1skin, 1 small intestine, 1 spleen, 1 stomach, 1 testis, 1 thymus, 1thyroid gland, 1 urinary bladder, 1 uterine cervix, 1 uterus, 1 vein;FIG. 2C) ALDH1L1, tissues from left to right: 1 adrenal gland, 1 artery,1 bone marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart,3 kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1skin, 1 small intestine, 1 spleen, 1 stomach, 1 testis, 1 thymus, 1thyroid gland, 1 urinary bladder, 1 uterine cervix, 1 uterus, 1 vein;FIG. 2D) FGG, tissues from left to right: 1 adrenal gland, 1 artery, 1bone marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1skin, 1 small intestine, 1 spleen, 1 stomach, 1 testis, 1 thymus, 1thyroid gland, 1 urinary bladder, 1 uterine cervix, 1 uterus, 1 vein;FIG. 2E) C8B, tissues from left to right: 1 adrenal gland, 1 artery, 1bone marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1skin, 1 small intestine, 1 spleen, 1 stomach, 1 testis, 1 thymus, 1thyroid gland, 1 urinary bladder, 1 uterine cervix, 1 uterus, 1 vein;and FIG. 2F) HSD17B6, tissues from left to right: including 1 adrenalgland, 1 artery, 1 bone marrow, 1 brain (whole), 1 breast, 1 colon, 1esophagus, 1 heart, 3 kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1lymph node, 1 ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivarygland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1stomach, 1 testis, 1 thymus, 1 thyroid gland, 1 urinary bladder, 1uterine cervix, 1 uterus, and 1 vein.

FIG. 3 shows exemplary flow cytometry results after peptide-specificmultimer staining. Further explanations see example 4.

FIG. 4 shows exemplary flow cytometry results after peptide-specificmultimer staining. Further explanations see example 4.

EXAMPLES Example 1 Identification and Quantitation of Tumor AssociatedPeptides Presented on the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from Universitätsklinik fürAllgemeine, Viszeralund Transplantationschirurgie, Tübingen, Germany;Istituto Nazionale Tumori “Pascale”. Molecular Biology and ViralOncology Unit, Via Mariano, Naples, Italy; Bio-Options Inc., Brea,Calif., USA; ProteoGenex Inc., Culver City, Calif., USA; AsterandEurope, Royston Herts, United Kingdom. Written informed consents of allpatients had been given before surgery. Tissues were shock-frozenimmediately after surgery and stored until isolation of TUMAPs at −70°C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk, K., 1991; Seeger, F. H. T., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, -B, -C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ—velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.2007a). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al. 2007b; Sturm et al. 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose HCC samples to a baseline of normal tissue samples.

Presentation profiles of exemplary over-presented peptides are shown inFIG. 1.

Presentation scores for exemplary peptides are shown in Table 8.

TABLE 8  Presentation scores. SEQ ID No. Sequence PeptidePresentation 1VMAPFTMTI +++ 2 KLQAGTVFV +++ 4 KLQDFSDQL +++ 5 ALVEQGFTV +++ 6KLSPTVVGL +++ 7 ALVDTLKFV +++ 8 KLLEEATISV + 9 ALANQKLYSV + 10SLLEEFDFHV +++ 11 SLSQELVGV + 12 FLAELAYDL +++ 14 ALADLTGTVV +++ 15LLYGHTVTV + 16 SLLGGNIRL ++ 17 RVAS*PTSGV + 19 FLEETKATV +++ 20KLSNVLQQV +++ 21 QLIEVSSPITL +++ 22 RIAGIRGIQGV +++ 23 RLYDPASGTISL + 24SLAEEKLQASV +++ 25 SLDGKAALTEL +++ 26 SLLHTIYEV +++ 27 TLPDFRLPEI +++ 28TLQDHLNSL +++ 29 YIQDEINTI +++ 30 YLGEGPRMV ++ 31 YQMDIQQEL ++ 32ALNAVRLLV +++ 33 LLHGHIVEL + 34 SLAEGTATV +++ 38 ALADVVHEA + 39ALDPKANFST +++ 40 ALLAEGITVVV + 42 ALLGGNVRMML +++ 44 ALQDAIRQL + 47FLDTPIAKV + 49 FLYPEKDEPT +++ 51 GLAEELVRA + 52 GLFNAELLEA + 53GLIHLEGDTV +++ 54 GLLDPNVKSIFV +++ 55 GLYGRTIEL + 56 GVLPGLVGV + 57HLTEAIQYV ++ 58 ILADLNLSV + 59 ILADTFIGV ++ 60 ILSPLSVAL + 61KIADFELPTI  +++ 62 KIAGTNAEV ++ 66 KLHEEIDRV ++ 67 KLKETIQKL +++ 70KLLDLETERILL ++ 71 KLLDNWDSV +++ 72 KLSEAVTSV + 75 KQMEPLHAV + 76LLADIGGDPFAA +++ 77 LLHEENFSV + 79 LLLSTGYEA +++ 81 NLASFIEQVAV ++ 82NVFDGLVRV + 83 QLHDFVMSL +++ 84 QLTPVLVSV ++ 85 RILPKVLEV + 86 RLAAFYSQV+++ 88 RLIDRIKTV +++ 89 RLIEEIKNV +++ 91 RLPDIPLRQV + 93 RLYTMDGITV  +++94 RMSDVVKGV +++ 96 SLLEEPNVIRV ++ 97 SLLPQLIEV ++ 98 SLLSPEHLQYL ++ 99SLSAFLPSL +++ 101 SLWEGGVRGV  +++ 103 SMGDHLVVVA  +++ 107 TLGQFYQEV +++108 TLLKKISEA +++ 109 TLYALSHAV + 111 TVMDIDTSGTFNV + 113 VLMDKLVEL ++114 VLSQVYSKV +++ 116 VVVIPAISAV +++ 117 YAFPKSITV +++ 119 YLDKNLTVSV +120 YLGEEYVKA +++ 121 YLITGNLEKL + 122 YLSQAADGAKVL +++ 123 YLWDLDHGFAGV++ 124 LLIDVVTYL +++ 126 TLLDSPIKV ++ 127 VLIGSNHSL + 128 GLAFSLNGV +129 SQADVIPAV + 130 ALDAGAVYTL ++ 131 ALDSGAFQSV ++ 132 ALHEEVVGV + 133ALLEM DARL + 134 ALLETNPYLL ++ 135 ALLGKIEKV + 137 ALPTVLVGV ++ 139ALSSKPAEV + 142 AVIGGLIYV ++ 144 FIQLITGV + 146 FLWTEQAHTV + 147GLAPGGLAVV + 148 GLFAPLVFL +++ 151 HLAKVTAEV + 154 KLTDHLKYV + 161RLLDEQFAV + 162 RLMSALTQV ++ 163 RLTESVLYL ++ 164 RMLIKLLEV + 167SLAESSFDV ++ 168 SLAVLVPIV + 169 SLFEWFHPL + 170 SLHNGVIQL + 171SLIPAVLTV + 172 SLLNFLQHL + 173 SLTSEIHFL + 174 TLAELGAVQV + 176TLGQIWDV + 177 VLDEPYEKV + 179 YIHNILYEV ++ 180 YLGPHIASVTL ++ 181YLLEKFVAV + 184 VVLDGGQIVTV + 185 ALFPALRPGGFQA ++ 186 VLLAQIIQV + 187SYPTFFPRF + 188 RYSAGWDAKF + 189 AFSPDSHYLLF +++ 190 RYNEKCFKL +++ 191KYPDIISRI ++ 192 SYITKPEKW + 193 IYPGAFVDL +++ 194 QYASRFVQL +++ 195RYAPPPSFSEF +++ 196 AYLKWISQI +++ 197 RWPKKSAEF + 198 LYWSHPRKF + 200AYLLQPSQF +++ 201 AYVNTFHNI +++ 202 AYGTYRSNF +++ 203 YYGILQEKI +++ 204KYRLTYAYF ++ 205 VYGLQRNLL + 206 KWPETPLLL +++ 208 SYNPAENAVLL ++ 210AYPAIRYLL ++ 211 IYIPSYFDF ++ 212 VYGDVISNI +++ 213 YYNKVSTVF + 214IYVTSIEQI +++ 217 DYIPYVFKL +++ 218 VYQGAIRQI + 219 GVMAGDIYSV + 220SLLEKELESV ++ 221 ALCEENMRGV + 224 ALASVIKEL + 225 KMDPVAYRV + 226AVLGPLGLQEV + 227 ALLKVNQEL + 228 YLITSVELL ++ 229 KMFESFIESV ++ 230VLTEFTREV + 231 RLFNDPVAMV ++ 233 ALLGKLDAI + 234 YLEPYLKEV + 236ALADKELLPSV ++ 237 ALRGEIETV +++ 238 AMPPPPPQGV ++ 239 FLLGFIPAKA + 240FLWERPTLLV +++ 241 FVLPLLGLHEA ++ 242 GLFAPVHKV + 243 GLLDNPELRV +++ 244KIAELLENV + 245 KLGAVFNQV + 248 KLNDLIQRL + 249 LLLGERVAL +++ 250NLAEVVERV ++ 251 RLFADILNDV ++ 252 RTIEYLEEV + 253 RVPPPPQSV + 255SLFGQDVKAV +++ 256 SLFQGVEFHYV + 257 SLLEKAGPEL +++ 258 SLMGPVVHEV + 260TLMDMRLSQV ++ 261 VLFQEALWHV ++ 263 VLYPSLKEI + 264 VMQDPEFLQSV ++ 265WLIEDGKVVTV ++ 266 SLLESNKDLLL + 267 ALNENINQV + 268 KLYQEVEIASV + 269YLMEGSYNKV + 270 SVLDQKILL ++ 271 LLLDKLILL + 272 QQLDSKFLEQV + 273AILETAPKEV ++ 274 ALAEALKEV + 275 ALIEGAGILL ++ 276 ALLEADVNIKL + 277ALLEENSTPQL + 278 ALTSVVVTL + 279 ALWTGMHTI + 281 GLLAGDRLVEV + 282GQFPSYLETV ++ 283 ILSGIGVSQV + 284 KLDAFVEGV + 286 KVLDKVFRA + 288LLDDSLVSI + 289 LLLEEGGLVQV ++ 290 NLIDLDDLYV ++ 292 RIPAYFVTV + 293FLASESLIKQI ++ 295 SLFSSPPEI ++ 297 TLFYSLREV + 298 TMAKESSIIGV ++ 299ALLRVTPFI + 301 VLADFGARV +++ 302 KIQEILTQV +++ 303 GVYDGEEHSV + 304SLIDQFFGV +++ 305 GVLENIFGV + 308 ALLRTVVSV + 309 GLIEIISNA + 310SLWGGDVVL + 311 FLIPIYHQV + 312 RLGIKPESV +++ 313 LTAPPEALLMV + 314YLAPFLRNV + 315 KVLDGSPIEV + 316 LLREKVEFL + 317 KLPEKWESV ++ 318KLNEINEKI + 319 KLFNEFIQL + 320 GLADNTVIAKV + 322 ILYDIPDIRL + 324RLFETKITQV ++ 326 ALSDGVHKI ++ 327 GLNEEIARV ++ 328 RLEEDDGDVAM + 329SLIEDLILL +++ 330 SMSADVPLV ++ 332 AMLAVLHTV + 334 SILTIEDGIFEV + 335SLLPVDIRQYL ++ 336 YLPTFFLTV + 337 TLLAAEFLKQV + 338 KLFDSDPITVTV +++340 KVFDEVIEV + 342 AMSSKFFLV + 343 LLLPDYYLV + 345 SYNPLWLRI +++ (A*24)346 LYQILQGIVF +++ (A*24) 347 ALNPADITV + The table lists peptides thatare very highly over-presented on tumors compared to a panel of normaltissues (+++), highly over-presented on tumors compared to a panel ofnormal tissues (++) or over-presented on tumors compared to a panel ofnormal tissues (+). S* = phosphoserine

Example 2 Expression Profiling of Genes Encoding the Peptides of theInvention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalization, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0. Exemplary expression profiles of source genes of the presentinvention that are highly over-expressed or exclusively expressed in HCCare shown in FIG. 2. Expression scores for further exemplary genes areshown in Table 9.

TABLE 9  Expression scores. Gene SEQ ID No Sequence Expression 1VMAPFTMTI +++ 2 KLQAGTVFV ++ 3 ILDDNMQKL + 4 KLQDFSDQL +++ 5 ALVEQGFTV+++ 7 ALVDTLKFV +++ 10 SLLEEFDFHV + 13 GLIDTETAMKAV +++ 19 FLEETKATV +++20 KLSNVLQQV +++ 21 QLIEVSSPITL +++ 25 SLDGKAALTEL +++ 27 TLPDFRLPEI +++28 TLQDHLNSL +++ 29 YIQDEINTI +++ 31 YQMDIQQEL +++ 38 ALADVVHEA + 39ALDPKANFST + 41 ALLELDEPLVL +++ 42 ALLGGNVRMML + 44 ALQDAIRQL + 45ALQDQLVLV ++ 46 AMAEMKWL ++ 48 FLLEQPEIQV + 49 FLYPEKDEPT +++ 50FTIPKLYQL +++ 52 GLFNAELLEA +++ 53 GLIHLEGDTV +++ 55 GLYGRTIEL +++ 60ILSPLSVAL + 61 KIADFELPTI +++ 62 KIAGTNAEV + 66 KLHEEIDRV +++ 67KLKETIQKL +++ 68 KLLAATVLLL +++ 73 KLTLVIISV +++ 74 KLYDLELIV +++ 76LLADIGGDPFAA + 81 NLASFIEQVAV + 82 NVFDGLVRV +++ 83 QLHDFVMSL +++ 84QLTPVLVSV ++ 85 RILPKVLEV ++ 87 RLFEENDVNL +++ 90 RLLDVLAPLV + 93RLYTMDGITV +++ 94 RMSDWKGV + 95 SICNGVPMV ++ 97 SLLPQLIEV +++ 100SLVGDIGNVNM +++ 103 SMGDHLWVA + 105 SVYDGKLLI + 106 TLAAIIHGA ++ 107TLGQFYQEV +++ 109 TLYALSHAV +++ 110 TVGGSEILFEV +++ 113 VLMDKLVEL +++114 VLSQVYSKV +++ 116 WVIPAISAV ++ 117 YAFPKSITV + 119 YLDKNLTVSV ++ 120YLGEEYVKA +++ 124 LLIDVVTYL +++ 126 TLLDSPIKV +++ 129 SQADVIPAV ++ 130ALDAGAVYTL ++ 132 ALHEEVVGV ++ 141 AMGEKSFSV + 142 AVIGGLIYV +++ 145FLIAEYFEHV ++ 146 FLWTEQAHTV ++ 148 GLFAPLVFL + 149 GLLSGLDIMEV +++ 154KLTDHLKYV +++ 157 QLLPNLRAV + 158 RIISGLVKV ++ 160 RLLAKIICL +++ 163RLTESVLYL ++ 165 RVIEHVEQV ++ 168 SLAVLVPIV +++ 172 SLLNFLQHL + 173SLTSEIHFL + 175 TLFEHLPHI ++ 177 VLDEPYEKV ++ 182 YLLHFPMAL +++ 183YLYNNEEQVGL +++ 187 SYPTFFPRF + 188 RYSAGWDAKF +++ 192 SYITKPEKW + 193IYPGAFVDL + 200 AYLLQPSQF +++ 204 KYRLTYAYF +++ 206 KWPETPLLL + 215IYTGNISSF +++ 217 DYIPYVFKL +++ 218 VYQGAIRQI +++ 228 YLITSVELL + 233ALLGKLDAI + 249 LLLGERVAL + 255 SLFGQDVKAV + 259 TLITDGMRSV + 263VLYPSLKEI + 273 AILETAPKEV + 275 ALIEGAGILL + 286 KVLDKVFRA + 296SLLSGRISTL + 298 TMAKESSIIGV + 301 VLADFGARV ++ 302 KIQEILTQV + 315KVLDGSPIEV ++ 318 KLNEINEKI +++ 320 GLADNTVIAKV + 324 RLFETKITQV ++ 327GLNEEIARV + 336 YLPTFFLTV + 341 YLAIGIHEL ++ 345 SYNPLWLRI (A*24) ++ Thetable lists peptides from genes that are very highly overexpressed intumors compared to a panel of normal tissues (+++), highly overexpressedin tumors compared to a panel of normal tissues (++) or overexpressed intumors compared to a panel of normal tissues (+).

Example 3 UV-Ligand Exchange/Peptide Binding to HLA-A*02 and HLA-A*24

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko B,Toebes M, Hadrup S R, van Esch W J, Molenaar A M, Schumacher T N, OvaaH. Generation of peptide-MHC class I complexes through UV-mediatedligand exchange. Nat Protoc. 2006; 1(3):1120-32).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*0201/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH2SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 10A MHC class I binding scores Seq ID Peptide code Peptideexchange 12 GPC3-001 ++++ 5 APOB-001 ++++ 7 APOB-002 ++++ 1 APOB-003++++ 13 HSD11B1-001 ++++ 227 SAMM-001 ++++ 4 APOB-004 ++++ 232MAPKAPK5-001 ++++ 10 USO-001 ++++ 304 USP14-001 ++++ 219 ADF-012 ++++223 IDI1-001 ++++ 224 IFT81-001 ++++ 14 NCST-001 ++++ 228 ACSL4-001 ++++230 IPO9-001 ++++ 15 SLC35B2-001 ++++ 16 ACSL3-001 ++++ 303 MAGEB2-001+++ 226 THT-001 +++ 8 DYM-001 +++ 6 AXIN2-001 +++ 225 QAR-001 +++ 2ALDH1L1-001 +++ 221 EEF2-001 +++ 220 DRG2-001 +++ 301 C1QTNF3-001 +++ 11ZNF318-001 +++ <20% = +; 20%-49% = ++; 50%-75% = +++; >=75% = ++++

TABLE 10B  MHC class I binding scores SEQ ID No SequencePeptide exchange 1 VMAPFTMTI “++++” 2 KLQAGTVFV “+++” 3 ILDDNMQKL “+++”4 KLQDFSDQL “++++” 5 ALVEQGFTV “++++” 6 KLSPTVVGL “+++” 7 ALVDTLKFV“++++” 8 KLLEEATISV “+++” 9 ALANQKLYSV “+++” 10 SLLEEFDFHV “++++” 11SLSQELVGV “+++” 12 FLAELAYDL “++++” 13 GLIDTETAMKAV “++++” 14 ALADLTGTVV“++++” 15 LLYGHTVTV “++++” 16 SLLGGNIRL “++++” 17 RVAS*PTSGV “+++” 18ALYGKTEVV “+++” 19 FLEETKATV “+++” 20 KLSNVLQQV “++++” 21 QLIEVSSPITL“++++” 22 RIAGIRGIQGV “++++” 23 RLYDPASGTISL “+++” 24 SLAEEKLQASV “+++”25 SLDGKAALTEL “+++” 26 SLLHTIYEV “+++” 27 TLPDFRLPEI  “++++” 28TLQDHLNSL “+++” 29 YIQDEINTI “+++” 30 YLGEGPRMV “+++” 31 YQMDIQQEL “+++”32 ALNAVRLLV “++++” 33 LLHGHIVEL “+++” 34 SLAEGTATV “+++” 35 SLQESILAQV“+++” 36 ILNVDGLIGV “+++” 37 LLLPLLPPLSP “+++” 38 ALADVVHEA “+++” 39ALDPKANFST “+++” 40 ALLAEGITWV “+++” 41 ALLELDEPLVL “++++” 42ALLGGNVRMML “+++” 43 ALLGVWTSV “++” 44 ALQDAIRQL “++++” 45 ALQDQLVLV“++++” 46 AMAEMKVVL “+++” 47 FLDTPIAKV “++” 48 FLLEQPEIQV “+++” 49FLYPEKDEPT “++” 50 FTIPKLYQL “++” 51 GLAEELVRA “++” 52 GLFNAELLEA “+++”53 GLIHLEGDTV “+++” 54 GLLDPNVKSIFV “+++” 55 GLYGRTIEL “+++” 56GVLPGLVGV “+++” 57 HLTEAIQYV “+++” 58 ILADLNLSV “++++” 59 ILADTFIGV“+++” 60 ILSPLSVAL “++++” 61 KIADFELPTI “++++” 62 KIAGTNAEV “++” 63KIDEKNFVV “+++” 64 KILEETLYV “+++” 65 KLFSGDELLEV “+++” 66 KLHEEIDRV“+++” 67 KLKETIQKL “+++” 68 KLLAATVLLL “++” 69 KLLDEVTYLEA “++++” 70KLLDLETERILL “++++” 71 KLLDNWDSV “++++” 72 KLSEAVTSV “+++” 74 KLYDLELIV“+++” 75 KQMEPLHAV “++” 76 LLADIGGDPFAA “+++” 77 LLHEENFSV “+++” 78LLIDDEYKV “+++” 80 LLYEGKLTL “++++” 81 NLASFIEQVAV “+++” 82 NVFDGLVRV“+++” 83 QLHDFVMSL “++++” 84 QLTPVLVSV “+++” 85 RILPKVLEV “+++” 86RLAAFYSQV “+++” 87 RLFEENDVNL “+++” 88 RLIDRIKTV “++” 89 RLIEEIKNV “++”90 RLLDVLAPLV “+++” 91 RLPDIPLRQV “+++” 92 RLPPDTLLQQV “+++” 93RLYTMDGITV “++” 94 RMSDVVKGV “+++” 95 SICNGVPMV “+++” 96 SLLEEPNVIRV“++++” 97 SLLPQLIEV “++++” 98 SLLSPEHLQYL “+++” 99 SLSAFLPSL “++++” 100SLVGDIGNVNM “++” 101 SLWEGGVRGV “+++” 102 SLWSVARGV “+++” 103 SMGDHLWVA“+++” 104 SVWFGPKEV “+++” 105 SVYDGKLLI “++++” 106 TLAAIIHGA “+++” 107TLGQFYQEV “++++” 108 TLLKKISEA “+++” 109 TLYALSHAV “+++” 110 TVGGSEILFEV“++++” 111 TVMDIDTSGTFNV “+++” 112 VLGEVKVGV “++++” 113 VLMDKLVEL “++++”114 VLSQVYSKV “+++” 115 VVLDDKDYFL “+++” 116 VVVIPAISAV “++++” 117YAFPKSITV “+++” 118 YLDDEKNWGL “+++” 119 YLDKNLTVSV “+++” 120 YLGEEYVKA“++” 121 YLITGNLEKL “+++” 122 YLSQAADGAKVL “++” 123 YLWDLDHGFAGV “+++”124 LLIDVVTYL “++++” 125 ALYGRLEVV “++++” 126 TLLDSPIKV “+++” 127VLIGSNHSL “++++” 128 GLAFSLNGV “++++” 129 SQADVIPAV “+++” 130 ALDAGAVYTL“++++” 131 ALDSGAFQSV “+++” 132 ALHEEVVGV “+++” 133 ALLEMDARL “+++” 134ALLETNPYLL “++++” 135 ALLGKIEKV “+++” 136 ALLNQHYQV “+++” 137 ALPTVLVGV“++++” 138 ALSQVTLLL “++++” 139 ALSSKPAEV “+++” 140 ALTSISAGV “++++” 141AMGEKSFSV “++++” 142 AVIGGLIYV “++++” 145 FLIAEYFEHV “++” 146 FLWTEQAHTV“++” 147 GLAPGGLAVV “+++” 148 GLFAPLVFL “++++” 149 GLLSGLDIMEV “++++”150 GLSNLGIKSI “++++” 151 HLAKVTAEV “+++” 152 KLDNNLDSV “+++” 154KLTDHLKYV “+++” 156 LLFPHPVNQV “++++” 157 QLLPNLRAV “+++” 158 RIISGLVKV“++” 159 RLFPDGIVTV “+++” 160 RLLAKIICL “++” 161 RLLDEQFAV “+++” 162RLMSALTQV- “++” 163 RLTESVLYL “+++” 164 RMLIKLLEV “+++” 165 RVIEHVEQV“++++” 166 SILDIVTKV “+++” 167 SLAESSFDV “+++” 168 SLAVLVPIV “+++” 169SLFEWFHPL “+++” 170 SLHNGVIQL “+++” 171 SLIPAVLTV “+++” 172 SLLNFLQHL“+++” 173 SLTSEIHFL “+++” 174 TLAELGAVQV  “+++” 175 TLFEHLPHI “+++” 176TLGQIWDV “++++” 177 VLDEPYEKV “+++” 178 YIFTTPKSV “+++” 179 YIHNILYEV“++++” 180 YLGPHIASVTL “+++” 181 YLLEKFVAV “+++” 182 YLLHFPMAL “+++” 183YLYNNEEQVGL “++” 184 VVLDGGQIVTV “+++” 185 ALFPALRPGGFQA “+++” 186VLLAQIIQV “+++” 187 SYPTFFPRF “++++” 188 RYSAGWDAKF “++++” 189AFSPDSHYLLF “+++” 190 RYNEKCFKL “++++” 191 KYPDIISRI “++++” 192SYITKPEKW “++++” 193 IYPGAFVDL “++++” 195 RYAPPPSFSEF  “++++” 196AYLKWISQI “++++” 197 RWPKKSAEF “++++” 198 LYWSHPRKF “++++” 199 KFVTVQATF“++++” 200 AYLLQPSQF “++++” 201 AYVNTFHNI “++++” 202 AYGTYRSNF “++++”203 YYGILQEKI “++++” 205 VYGLQRNLL “++++” 206 KWPETPLLL “++++” 207IYLERFPIF “++++” 208 SYNPAENAVLL “++++” 209 VFHPRQELI “+++” 210AYPAIRYLL “++++” 211 IYIPSYFDF “++++” 212 VYGDVISNI “++++” 213 YYNKVSTVF“++++” 214 IYVTSIEQI “++++” 215 IYTGNISSF “++++” 216 IYADVGEEF “++++”217 DYIPYVFKL “++++” 218 VYQGAIRQI “++++” 219 GVMAGDIYSV “++++” 220SLLEKELESV “+++” 221 ALCEENMRGV “+++” 222 LTDITKGV “++” 223 FLFNTENKLLL“++++” 224 ALASVIKEL “+++” 225 KMDPVAYRV “+++” 226 AVLGPLGLQEV “+++” 227ALLKVNQEL “++++” 228 YLITSVELL “++++” 229 KMFESFIESV “+++” 230 VLTEFTREV“++++” 231 RLFNDPVAMV “++++” 232 KLAEIVKQV “++++” 233 ALLGKLDAI “++++”234 YLEPYLKEV “++++” 235 KLFEEIREI “++++” 236 ALADKELLPSV “+++” 237ALRGEIETV “+++” 238 AMPPPPPQGV “++” 239 FLLGFIPAKA “+++” 240 FLWERPTLLV“+++” 241 FVLPLLGLHEA “++” 242 GLFAPVHKV “+++” 243 GLLDNPELRV “+++” 244KIAELLENV “++++” 245 KLGAVFNQV “++++” 246 KLISSYYNV “+++” 247KLLDTMVDTFL “++++” 248 KLNDLIQRL “+++” 249 LLLGERVAL “++++” 250NLAEVVERV “++++” 251 RLFADILNDV  “++++” 252 RTIEYLEEV “+++” 253RVPPPPQSV “++” 254 RVQEAIAEV “+++” 255 SLFGQDVKAV “+++” 256 SLFQGVEFHYV“+++” 257 SLLEKAGPEL “+++” 258 SLMGPVVHEV “+++” 259 TLITDGMRSV- “++” 260TLMDMRLSQV “+++” 261 VLFQEALWHV “+++” 262 VLPNFLPYNV “+++” 263 VLYPSLKEI“+++” 264 VMQDPEFLQSV “++++” 265 WLIEDGKVVTV “++++” 266 SLLESNKDLLL“+++” 267 ALNENINQV “+++” 268 KLYQEVEIASV “++++” 269 YLMEGSYNKV “+++”270 SVLDQKILL “+++” 271 LLLDKLILL “++++” 272 QQLDSKFLEQV “+++” 273AILETAPKEV “+++” 274 ALAEALKEV “++++” 275 ALIEGAGILL “+++” 276ALLEADVNIKL “++++” 277 ALLEENSTPQL “+++” 278 ALTSVVVTL “++++” 279ALWTGMHTI “+++” 280 ATLNIIHSV “+++” 281 GLLAGDRLVEV “+++” 282 GQFPSYLETV“+++” 283 ILSGIGVSQV “+++” 284 KLDAFVEGV “+++” 285 KLLDLSDSTSV “+++” 286KVLDKVFRA “++++” 287 LIGEFLEKV “++++” 288 LLDDSLVSI “+++” 290 NLIDLDDLYV“++++” 291 QLIDYERQL “+++” 292 RIPAYFVTV “++” 293 FLASESLIKQI “++” 294RLIDLHTNV “++++” 295 SLFSSPPEI “+++” 296 SLLSGRISTL “+++” 297 TLFYSLREV“+++” 298 TMAKESSIIGV “++” 299 ALLRVTPFI “+++” 300 TLAQQPTAV “++” 302KIQEILTQV “++++” Binding of HLA-class I restricted peptides to HLA-A*02or HLA-A*24 depending frompeptide sequence was classified by peptideexchange yield: ≧10% = +; ≧20% = ++; ≧50 = +++; ≧75% = ++++. S* =phosphoserine.

Example 4 In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for 22 HLA-A*0201 restricted TUMAPsof the invention so far, demonstrating that these peptides are T-cellepitopes against which CD8+ precursor T cells exist in humans (Table11).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV from modified Melan-A/MART-1) andA*0201/DDX5-001 (YLLPAIVHI from DDX5), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oreg., USA). In vitro priming of specific multimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for HCC Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for threepeptides of the invention are shown in FIG. 3 and FIG. 4 together withcorresponding negative controls. Results for 22 peptides from theinvention are summarized in Table 11A.

TABLE 11A in vitro immunogenicity of HLA class I peptides of theinvention Seq ID Peptide ID wells donors 225 QAR-001 +++ ++++ 1 APOB-003++ ++++ 2 ALDH1L1-001 ++ ++++ 301 C1QTNF3-001 ++ ++++ 15 SLC35B2-001 ++++++ 16 ACSL3-001 ++ ++++ 12 GPC3-001 + ++++ 7 APOB-002 + ++++ 303MAGEB2-001 + ++ 227 SAMM-001 + +++ 4 APOB-004 + ++++ 226 THT-001 + ++++6 AXIN2-001 + ++ 232 MAPKAPK5-001 + +++ 10 USO-001 + ++ 304 USP14-001 +++++ 219 ADF-012 + ++++ 224 IFT81-001 + +++ 11 ZNF318-001 + ++ 14NCST-001 + ++ 228 ACSL4-001 + ++ 230 IP09-001 + ++++ Exemplary resultsof in vitro immunogenicity experiments conducted by the applicant forthe peptides of the invention. <20% = +; 20%-49% = ++; 50%-69% =+++; >=70% = ++++

TABLE 11B  in vitro immunogenicity of additional HLA class Ipeptides of the invention Wells Donors SEQ ID No Sequence positive [%]positive [%] 187 SYPTFFPRF “+” “++++” 189 AFSPDSHYLLF “+” “++” 190RYNEKCFKL “+” “++” 191 KYPDIISRI “+” “++++” 192 SYITKPEKW “+” “+++” 193IYPGAFVDL “+” “+++” 194 QYASRFVQL “+” “+++” 196 AYLKWISQI “+” “+++” 197RWPKKSAEF ++” “++” 198 LYWSHPRKF “+” “++” 199 KFVTVQATF “+” “++” 201AYVNTFHNI “++” “++” 202 AYGTYRSNF “+” “++++” 203 YYGILQEKI “+” “+++” 205VYGLQRNLL “+” “+++” 207 IYLERFPIF “++” “++++” 208 SYNPAENAVLL “+” “+++”209 VFHPRQELI “++” “++++” 210 AYPAIRYLL “++” “++++” 211 IYIPSYFDF “++”“++++” 212 VYGDVISNI “++” “++++” 215 IYTGNISSF “+” “++” 216 IYADVGEEF“+” “++” 217 DYIPYVFKL “+++” “++++” 218 VYQGAIRQI “+” “+++” Exemplaryresults of in vitro immunogenicity experiments conducted by theapplicant for HLA-A*24 restricted peptides of the invention. Results ofin vitro immunogenicity experiments are indicated. Percentage ofpositive wells and donors (among evaluable) are summarized as indicated1-20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++

Exemplary Results of Peptide-Specific In Vitro CD8+ T Cell Responses ofa Healthy HLA-A*02+Donor (FIG. 3)

CD8+ T cells were primed using artificial APCs coated with anti-CD28 mAband HLA-A*02 in complex with IMA-APOB-002 (Seq ID No 7) peptide (A,right panel) or IMA-APOB-003 (B, right panel, Seq ID No 1), orIMA-ALDH1L1-001 (C, right panel, Seq ID No 2), respectively. After threecycles of stimulation, the detection of peptide-reactive cells wasperformed by 2D multimer staining with A*02/APOB-002 (A) orA*02/APOB-003 (B), or A*02/ALDH1L1-001. Left panels (A, B, C) showcontrol staining of cells stimulated with irrelevant A*02/peptidecomplexes. Viable singlet cells were gated for CD8+ lymphocytes. Booleangates helped excluding false-positive events detected with multimersspecific for different peptides. Frequencies of specific multimer+ cellsamong CD8+ lymphocytes are indicated.

Exemplary Results of Peptide-Specific In Vitro CD8+ T Cell Responses ofa Healthy HLA-A*24+Donor (FIG. 4)

CD8+ T cells were primed using artificial APCs coated with anti-CD28 mAband HLA-A*24 in complex with IMA-KLHL24-001 (Seq ID No 190) peptide (A,right panel) or IMA-APOB-006 (B, right panel, Seq ID No 218),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*24/KLHL24-001 (A) or A*24/APOB-006 (B). Left panels (A and B) showcontrol staining of cells stimulated with irrelevant A*24/peptidecomplexes. Viable singlet cells were gated for CD8+ lymphocytes. Booleangates helped excluding false-positive events detected with multimersspecific for different peptides. Frequencies of specific multimer+ cellsamong CD8+ lymphocytes are indicated.

Example 5 Syntheses of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

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1. A peptide comprising an amino acid sequence selected from the groupof SEQ ID No. 1 to SEQ ID No. 300, and/or a variant sequence thereofwhich is at least 88% homologous to SEQ ID No. 1 to SEQ ID No. 300, andwherein said variant binds to MHC and/or induces T cells cross-reactingwith said variant peptide; and/or a pharmaceutical acceptable saltthereof, wherein said peptide, variant and/or salt is not a full-lengthpolypeptide and further wherein said peptide, variant or salt isoptionally modified and/or includes non-peptide bonds.
 2. The peptide,variant and/or salt according to claim 1, wherein said peptide has theability to bind to a molecule of the human major histocompatibilitycomplex (MHC) class-I or -II, and wherein said peptide, when bound toMHC, is capable of being recognized by CD4 and/or CD8 T cells.
 3. Thepeptide and/or variant and/or salt thereof according to claim 1, whereinthe amino acid sequence thereof comprises a continuous stretch of aminoacids according the group of SEQ ID No. 1 to SEQ ID No.
 300. 4. Thepeptide and/or variant and/or salt thereof according to claim 1, whereinsaid peptide or variant or salt thereof has an overall length of from 8to 100, optionally from 8 to 30, and optionally from 8 to 16 aminoacids, and optionally wherein the peptide consists or consistsessentially of an amino acid sequence according to SEQ ID No. 1 to SEQID No.
 300. 5. The peptide or variant and/or salt thereof according toany of claim 1, wherein said peptide is modified and/or includesnon-peptide bonds.
 6. The peptide or variant or salt thereof accordingto claim 1, wherein said peptide is part of a fusion protein, optionallycomprising N-terminal amino acids of the HLA-DR antigen-associatedinvariant chain (Ii).
 7. A nucleic acid, encoding a peptide or variantor salt thereof according to claim 1, optionally linked to aheterologous promoter sequence.
 8. An expression vector capable ofexpressing the nucleic acid according to claim
 7. 9. The peptide orvariant or salt thereof according to claim 1, capable of being used inmedicine.
 10. A host cell comprising the peptide, variant or saltaccording to claim 1, wherein said host cell preferably is an antigenpresenting cell optionally comprising a dendritic cell.
 11. A method forproducing the peptide or variant thereof according to claim 1, themethod comprising culturing a host cell that presents the peptide orvariant thereof, and isolating the peptide or variant or salt thereoffrom the host cell or a culture medium.
 12. An in vitro method forproducing activated T lymphocytes, the method comprising contacting invitro one or more T cells with antigen loaded human class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor an artificial construct mimicking an antigen-presenting cell for aperiod of time sufficient to activate said T cells in an antigenspecific manner, wherein said antigen is a peptide, variant or saltaccording to claim
 1. 13. An activated T cell, produced by the methodaccording to claim 12, that selectively recognizes a cell which presentsa polypeptide comprising an amino acid sequence that is at least 88%homologous to SEQ ID No. 1 to SEQ ID No.
 300. 14. A method for killingtarget cells in a patient which target cells present a polypeptidecomprising an amino acid sequence that is at least 88% homologous to SEQID No. 1 to SEQ ID No. 300, the method comprising administering to thepatient an effective number of activated T cells as defined in claim 13.15. An antibody, soluble or membrane-bound, that specifically recognizesthe peptide or variant or salt thereof according to claim 1, optionallythe peptide or variant or salt thereof is bound to an MHC molecule andoptionally the antibody carries a further effector function optionallycomprising an immune stimulating domain or toxin.
 16. A peptide, variantor salt according to claim 1 capable of being used for treatment ofcancer and/or in the manufacture of a medicament against cancer.
 17. Thepeptide, variant or salt according to claim 16, wherein said cancer isselected from the group of HCC, brain cancer, kidney cancer, pancreaticcancer, colon or rectal cancer or leukemia and other tumors that show anoverexpression of most preferably APOB, FASN, COPA, preferably GLUL,GPAM, PLIN2, SLC16A1, SLC9A3R1, PCBD1, SEC16A, AKR1C4, ABCB11, HAL,CYP2E1, C4A, C4B, ALDH1L1, CRP, ACSL4, EEF2, HLTF, FBXO22, GALK1, TMCO1,TMEM33, ZNF318, IPO9, AMACR, C1QTNF3, CYP4F8, CYP4F3, CYP4F11, CYP4F12,CYP4F2, MOCOS, A1CF, COL18A1, HPR, LBP, C19orf80, CFHR5, ITIH4, TMEM110,LARP4, LMF2, SLC10A5, SLC16A11, and still preferably ANKFY1, C12orf44,C16orf58, CPSF1, DCAF8, PEX19, DDX11, DDX12P, DECR2, NME4, DENND5B, DYM,EDC4, ERI3, FAM20A, FNDC3A, GPR107, GYG2, HEATR2, IFT81, KCTD3, SHKBP1,KIAA1324L, KLHL24, MARCH6, MBTPS2, MIR1279, CPSF6, NOC4L, NXF1, PANK2,PCNXL3, PIPSL, PSMD4, PSMD14, SLC35B1, TCP11L2, THNSL2, THOC2, TOMM5,TRAPPC6B, TRIM54, TRIM55, TRIM63, UGGT2, URB1, VPS54, WIZ, ZNF45, RFTN2,SCFD1, SERINC5, CCT7P2, CMAS, ANKS1A, C17orf70, CCT7, CDK5RAP2, CLPTM1,and another protein from which a peptide SEQ ID No. 1 to SEQ ID No. 300is derived from.
 18. A kit comprising: (a) a container comprising apharmaceutical composition containing the peptide(s) or the variant orsalt according to claim 1 in solution or in lyophilized form; (b)optionally, a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; (c) optionally, at least onemore peptide selected from the group consisting of SEQ ID No. 1 to SEQID No. 346, and (d) optionally, instructions for (i) use of the solutionor (ii) reconstitution and/or use of the lyophilized formulation. 19.The kit according to claim 18, further comprising one or more of (iii) abuffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.20. The kit according to claim 18, wherein said peptide is selected fromthe group consisting SEQ ID No. 1 to SEQ ID No.
 300. 21. A method forproducing a personalized anti-cancer vaccine for a compound-based and/orcellular therapy for an individual patient, said method comprising: a)identifying tumor-associated peptides (TUMAPs) presented by a tumorsample from the individual patient; b) comparing the peptides asidentified in a) with a warehouse of peptides that have been prescreenedfor immunogenicity and/or over-presentation in tumors as compared tonormal tissues c) selecting at least one peptide from the warehouse thatmatches a tumor-associated peptide identified in the patient; and d)manufacturing the personalized vaccine or compound-based or cellulartherapy product based on step c).
 22. The method according to claim 21,wherein said TUMAPs are identified by: a1) comparing expression datafrom the tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor.
 23. The method according to claim 21,wherein the sequences of MHC ligands are identified by eluting boundpeptides from MHC molecules isolated from the tumor sample, andsequencing the eluted ligands.
 24. The method according to claim 21,wherein the normal tissue corresponding to the tissue type of the tumorsample is obtained from the same patient.
 25. The method according toclaim 21, wherein the peptides included in the warehouse are identifiedbased on the following steps: aa. Performing genome-wide messengerribonucleic acid (mRNA) expression analysis by highly parallel methods,optionally comprising microarrays or sequencing-based expressionprofiling, comprising identify genes that over-expressed in a malignanttissue, compared with a normal tissue or tissues; ab. Selecting peptidesencoded by selectively expressed or over-expressed genes as detected instep aa, and ac. Determining an induction of in vivo T-cell responses bythe peptides as selected comprising in vitro immunogenicity assays usinghuman T cells from healthy donors or said patient; or ba. IdentifyingHLA ligands from said tumor sample using mass spectrometry; bb.Performing genome-wide messenger ribonucleic acid (mRNA) expressionanalysis by highly parallel methods, optionally comprising microarraysor sequencing-based expression profiling, comprising identify genes thatover-expressed in a malignant tissue, compared with a normal tissue ortissues; bc. Comparing the identified HLA ligands to said geneexpression data; bd. Selecting peptides encoded by selectively expressedor over-expressed genes as detected in step bc; be. Re-detecting ofselected TUMAPs from step bd on tumor tissue and lack of or infrequentdetection on healthy tissues and confirming the relevance ofover-expression at the mRNA level; and bf. Determining an induction ofin vivo T-cell responses by the peptides as selected comprising in vitroimmunogenicity assays using human T cells from healthy donors or saidpatient.
 26. The method according to claim 21, wherein theimmunogenicity of the peptides included in the warehouse is determinedby a method comprising in vitro immunogenicity assays, patientimmunomonitoring for individual HLA binding, MHC multimer staining,ELISPOT assays and/or intracellular cytokine staining.
 27. The methodaccording to claim 21, wherein said warehouse comprises a plurality ofpeptides selected from the group consisting of SEQ ID No. 1 to SEQ IDNo.
 346. 28. The method according to claim 21, further comprisingidentifying at least one mutation that is unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andselecting a peptide that correlates with the mutation for inclusion inthe vaccine or for the generation of cellular therapies.
 29. The methodaccording to claim 28, wherein said at least one mutation is identifiedby whole genome sequencing.
 30. A T-cell receptor, soluble ormembrane-bound, that is reactive with an HLA ligand, wherein said ligandhas at least 75% identity to an amino acid sequence selected from thegroup consisting of SEQ ID No. 1 to SEQ ID No.
 300. 31. The T-cellreceptor according to claim 30, wherein said amino acid sequence is atleast 88% identical to SEQ ID No. 1 to SEQ ID No.
 300. 32. The T-cellreceptor according to claim 30, wherein said amino acid sequenceconsists any of SEQ ID No. 1 to SEQ ID No.
 300. 33. The T-cell receptoraccording to claim 30, wherein said T-cell receptor is provided as asoluble molecule and optionally carries a further effector functionoptionally comprising an immune stimulating domain or toxin.
 34. Anucleic acid, encoding for a TCR according to claim 30, optionallylinked to a heterologous promoter sequence.
 35. An expression vectorcapable of expressing the nucleic acid according to claim
 34. 36. A hostcell comprising the nucleic acid according to claim 34, wherein saidhost cell optionally is a T cell or NK cell.
 37. A method for producingthe T cell receptor according to claim 30, said method comprisingculturing a host cell, and isolating said T cell receptor from said hostcell and/or a culture medium.
 38. A pharmaceutical compositioncomprising at least one active ingredient selected from the groupconsisting of a) a peptide selected from the group consisting of SEQ IDNo. 1 to SEQ ID No. 300; b) a T-cell receptor reactive with a peptideand/or the peptide-MHC complex according to a); c) a fusion proteincomprising a peptide according to a), and the N-terminal amino acids 1to 80 of the HLA-DR antigen-associated invariant chain (Ii); d) anucleic acid encoding for any of a) to c) or an expression vectorcomprising said nucleic acid, e) a host cell comprising the expressionvector of d, f) an activated T-lymphocyte, obtained by a methodcomprising contacting in vitro T cells with a peptide according to a)expressed on the surface of a suitable antigen-presenting cell for aperiod of time sufficient to activate said T cell in an antigen specificmanner, as well as a method to transfer these activated T cells into theautologous or other patients; g) an antibody, or soluble T-cellreceptor, reactive to a peptide and/or the peptide—MHC complex accordingto a) and/or a cell presenting a peptide according to a), andpotentially modified by fusion with for example immune-activatingdomains or toxins, h) an aptamer recognizing a peptide selected from thegroup consisting of SEQ ID No. 1 to SEQ ID No. 300 and/or a complex of apeptide selected from the group consisting of SEQ ID No. 1 to SEQ ID No.300 with a MHC molecule, i) a conjugated or labelled peptide or scaffoldaccording to any of a) to h) and a pharmaceutically acceptable carrier,and optionally, pharmaceutically acceptable excipients and/orstabilizers.
 39. An aptamer that specifically recognizes the peptide orvariant or salt thereof according to claim 1, optionally the peptide orvariant or salt thereof is bound to an MHC molecule.
 40. Apharmaceutical composition according to claim 38, comprising at leastone peptide, and optionally all peptides, selected from SEQ ID NOs 1, 2,7, 225, 228, 301, 303 and 312.